Research Report 2004 Research Report 2004 (Covers the Period 2002-2003)

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MDC Berlin-Buch

Max Delbrück Center for Molecular Medicine

Research Report 2004 Research Report 2004 (covers the period 2002-2003)

Max Delbrück Center for Molecular Medicine (MDC) Berlin-Buch

Robert-Rössle-Str. 10 13125 Berlin Germany

Tel.: +49-30-9406-0 Fax: + 49-30-949-4161 e-mail: [email protected]

This Research Report is also available on the World Wide Web http://www.mdc-berlin.de

The MDC is a member of the Helmholtz Association of National Research Centers (HGF)

The following Research Reports have been published previously: Research Report 1996 (covers the period 1992-1995) Research Report 1996/97 (covers the period 1996-1997) Research Report 2000 (covers the period 1998-1999) Research Report 2002 (covers the period 2000-2001)

Editor-in-Chief Pamela Cohen

Editorial Board Christian Alecke Barbara Bachtler Walter Birchmeier Carmen Birchmeier Udo Heinemann Helmut Kettenmann Achim Leutz Thomas Willnow

Coordination Brigitte Buntrock Sonja Giering Elisabeth Schmeitzner

Book Design Legend to Cover Figure: Hoch Drei GmbH, 10963 Berlin Propagation of DNA replication (this report page 46) Printing Reprinted from Molecular Cell, Vol. 10, Sporbert et al., Brandenburgische Universitätsdruckerei DNA polymerase clamp shows little turnover at und Verlagsgesellschaft Potsdam mbH established replication sites but sequential de novo assembly at adjacent origin clusters. 1355-1365, Printed in Germany 2004 Copyright (2002), with permission from Elsevier. Research Report 2004

Covers the period 2002/2003 2

Content

Inhalt

Foreword Vorwort ...... 8

Cardiovascular and Metabolic Diseases Herz-Kreislauf- und Stoffwechselerkrankungen ...... 12

Hypertension, Vascular Disease, and Kidney Disease

Molecular Cardiovascular Research Thomas E. Willnow ...... 15

Molecular Biology and Genetics of Cardiovascular Diseases Detlev Ganten ...... 18

Medical Genomics Norbert Hübner ...... 20

Molecular Biology of Peptide Hormones Michael Bader ...... 22

Genetics, Nephrology, Hypertension, and Vascular Injury Friedrich C. Luft ...... 24

Control of Smooth Muscle Cell Function Maik Gollasch (Helmholtz Fellow) ...... 26

Molecular Mechanisms of the Metabolic Syndrome Jens Jordan (Helmholtz Fellow) ...... 28

Functional Characterization of Newly Identified Human Importin Proteins Matthias Köhler (Helmholtz Fellow) ...... 30

Obesity and Hypertension Arya M. Sharma ...... 31

Heart Disease

Cardiovascular Molecular Genetics Ludwig Thierfelder ...... 33

Genetic Disorders of the Cardiovascular System Brenda Gerull (Helmholtz Fellow) ...... 35

Myocardial Regeneration Rainer Dietz ...... 36

Myocardial Nuclear Receptors in Heart Failure Martin W. Bergmann (Helmholtz Fellow) ...... 38

Cardiovascular Magnetic Resonance Matthias G. Friedrich ...... 40

Molecular Muscle Physiology Ingo L. Morano ...... 42 3

Cell Polarity During Development of Drosophila and Zebrafish Salim Abdelilah-Seyfried ...... 44

Cell Biology of Cardiovascular Diseases M. Cristina Cardoso ...... 46

Immunology of Cardiovascular Diseases Gerd Wallukat ...... 49

Neuromuscular and Cardiovascular Cell Biology Michael Gotthardt ...... 51

Metabolic Diseases, Genetics, Genomics, and Bioinformatics

Bioinformatics Jens Reich ...... 53

Gene Mapping and Identification in Monogenic and Multifactorial Diseases Peter Nürnberg ...... 56

Cancer Research Krebsforschungsprogramm ...... 60

Signalling Pathways, Cell Biology, and Cancer

Epithelial Signal Transduction, Invasion, and Metastasis Walter Birchmeier ...... 66

Genetics of Tumor Progression and Metastasis Ulrike S. Ziebold (Helmholtz Fellow) ...... 69

Surgical Oncology Peter M. Schlag ...... 70

Molecular Genetics of Cell Differentiation & Tumorigenesis Achim Leutz ...... 73

Translational Control of Gene Expression Cornelis F. Calkhoven (Helmholtz Fellow) ...... 75

Signal Transduction in Tumor Cells Claus Scheidereit ...... 77

Antigen Receptor Signaling in Lymphocytes Daniel Krappmann (Helmholtz Fellow) ...... 80

Intracellular Proteolysis Thomas Sommer ...... 81

Regulation of Nuclear Transport Processes Katrin Stade (Helmholtz Fellow) ...... 83

Initiation of DNA Replication Manfred Gossen ...... 84

Evolution, Regulation, and Genetic Applications of Transposable Elements in Vertebrates Zoltán Ivics ...... 86 4

Structural and Functional Genomics

Structural Studies of Proteins and Nucleic Acids by X-ray Crystallography Udo Heinemann ...... 88

Computer Simulation of Biomolecular Structures, Dynamics, and Interactions Heinz Sklenar ...... 91

Nucleosides, Nucleotides, and Oligonucleotides Eckart Matthes ...... 93

Tumor Genetics Siegfried Scherneck ...... 95

Tumor Immunology

Differentiation and Growth Control in Lymphocyte Development and Immunopathogenesis Martin Lipp ...... 97

Biology of Hodgkin´s Lymphoma Bernd Dörken ...... 101

Mechanisms of Immune Evasion in Tumor Biology and in Herpes Virus Infections Armin Rehm (Helmholtz Fellow) ...... 104

Identification of Target Structures for the Development of Molecular Therapies in Malignant B-cell Disorders Ralf C. Bargou ...... 106

Cell-biological Determinants of Treatment Response and Prognosis in Acute Leukemias Wolf-Dieter Ludwig ...... 108

Role of Apoptosis and Senescence in Tumor Development and Treatment Responses Clemens A. Schmitt ...... 110

Clinical and Molecular Oncology Peter Daniel ...... 112

Molecular Immunotherapy Antonio Pezzutto ...... 114

Molecular Immunology and Gene Therapy Thomas Blankenstein ...... 116

Molecular Cell Biology and Gene Therapy Wolfgang Uckert ...... 118

Molecular and Cell Biology of Hematopoietic Cells Martin Zenke ...... 120

Cellular Immunology of Autoimmune Reactions Kirsten Falk Olaf Rötzschke ...... 123

Experimental Pharmacology Iduna Fichtner ...... 125 5

Function and Dysfunction of the Nervous System Funktion und Dysfunktion des Nervensystems ...... 128

Signalling Pathways in the Nervous System Pathophysiological Mechanisms of Neurological and Psychiatric Disorders Imaging of the Live Brain

Mouse Genetics – Tools for the Functional Analysis of Genes that are Important for Development and Disease Carmen Birchmeier ...... 131

Molecular Control of Spinal Cord and Peripheral Nervous System Development Stefan Britsch (Helmholtz Fellow) ...... 134

Genetic Analysis of Nervous System Development and Function Alistair N. Garratt (Helmholtz Fellow) ...... 136

Cellular Neurosciences Helmut Kettenmann ...... 138

Brain Energy Metabolism Susanne Arnold (Emmy Noether Research Group) ...... 141

Developmental Neurobiology Fritz G. Rathjen ...... 142

Growth Factors and Regeneration Gary Lewin ...... 144

Proteomics and Molecular Mechanisms of Neurodegenerative Disorders Erich Wanker ...... 146

Neurodegeneration Christiane Alexander ...... 149

Neuronal Stem Cells Gerd Kempermann ...... 151

Bioethics and Science Communication Christof Tannert ...... 153

Academics

Academic Appointments Berufungen ...... 156

Awards Preise ...... 159

Helmholtz Fellows Helmholtz-Stipendiaten ...... 160

PhD Program PhD-Programm ...... 161

Congresses and Scientific Meetings Kongresse und Wissenschaftliche Tagungen ...... 162

Seminars Seminare ...... 164 6

Structure and Organization Struktur und Organisation

The MDC Berlin-Buch Das MDC Berlin-Buch ...... 174

Clinical Research Die klinische Forschung ...... 174

The Helmholtz Association Die Helmholtz-Gemeinschaft ...... 176

The Campus Berlin-Buch Der Campus Berlin-Buch ...... 178

Organizational Structure Organisationsstruktur ...... 181

Supporting Divisions Stabsstellen ...... 185

Safety Sicherheit ...... 185

Building and Technology Bau und Technik ...... 186

Auditing, Legal Affairs, Patents, and Licenses Revision und Recht – Patente und Lizenzen ...... 187

Technology Transfer Technologietransfer ...... 187

Press and Public Relations Presse- und Öffentlichkeitsarbeit ...... 188

Administration Verwaltung ...... 190

Personnel Personal ...... 190

Finances Finanzen ...... 191

Purchasing and Materials Management Einkauf und Materialwirtschaft ...... 192 7

Central Facilities Zentrale Dienste ...... 193

Library Bibliothek ...... 193

Animal Facilities Tierhaltung ...... 194

Data and Image Processing EDV- und Bildverarbeitung ...... 195

Maintenance and General Services Haustechnik ...... 195

Index ...... 196

Organigram Organigramm ...... 204

Campus Map ...... Inside Back Cover Campusplan ...... Innenumschlag hinten

How to find your way to the MDC ...... Inside Back Cover Wie gelangen Sie zum MDC ...... Innenumschlag hinten 8

Foreword Vorwort

We are pleased to present the 2004 Research Report of the Wir präsentieren Ihnen hiermit den wissenschaftlichen Max Delbrück Center for Molecular Medicine (MDC) Ber- Bericht 2004 des Max-Delbrück-Centrums für Molekulare lin-Buch, which covers the research period of the years 2002 Medizin (MDC) Berlin-Buch, der die Forschungsperiode and 2003. Founded in 1992, the MDC is a young research 2002 und 2003 umfasst. Das MDC ist ein junges Forschungs- institute funded by the German Federal government (90%) institut, gegründet 1992, das vom Bund und dem Land Berlin and the State of Berlin (10%) and is a member of the Helm- im Verhältnis 90 zu 10 getragen wird und zur Helmholtz- holtz Association of National Research Centers (Helmholtz- Gemeinschaft Deutscher Forschungszentren (HGF) gehört. Gemeinschaft Deutscher Forschungszentren (HGF)). In ad- Das MDC ist für seine Forschung stark auf die Einwerbung dition to federal and state funds, the MDC augments its total von Drittmitteln angewiesen, die einen großen Teil seiner research budget via third party financial resources. Indeed, effektiven Forschungsmittel ausmachen. Wie erfolgreich das with a sum of 11.2 million Euros, the MDC was ranked third MDC dabei ist, zeigt sich daran, dass es bei der Einwerbung of non-university research institutions in procuring grants von Fördermitteln bei der Deutschen Forschungsgemeinschaft from the German Research Association (Deutsche For- (DFG) unter den außeruniversitären Forschungseinrichtun- schungsgemeinschaft (DFG)). Furthermore, the Institute for gen mit 11,2 Millionen Euro Platz drei belegt. Weiterhin hat Scientific Information (ISI/USA) National Citation Report die Analyse des Institute for Scientific Information (ISI/ placed the MDC among the top seven best molecular biologi- USA) im National Citation Report gezeigt, dass das MDC cal institutes in Germany. inzwischen zu den besten sieben molekularbiologischen Instituten Deutschlands gehört. Research at the MDC focuses on the molecular analysis and treatment of the most prevalent diseases in the population, Das MDC beschäftigt sich wissenschaftlich mit der moleku- namely cardiovascular diseases, cancer, and neurological laren Analyse und Therapie der wichtigsten Volkskrankhei- diseases. Two research clinics of the Charité University ten, Herz-Kreislauf-, Krebs- und neurologischen Erkrankun- Medical School in Berlin-Buch, the Franz Volhard Clinic for gen. Angeschlossen sind dem MDC zwei Forschungskliniken Cardiovascular Diseases as well as the Robert Rössle Cancer der Charité – Universitätsmedizin Berlin in Berlin-Buch, die Clinic, are connected to the MDC. Regarding patient care, Franz-Volhard-Klinik für Herzkreislauf-Erkrankungen sowie these two clinics are part of the Helios Clinics GmbH. The die Robert-Rössle-Krebsklinik. In der Krankenversorgung collaboration between the MDC and the Buch Hospitals in sind diese beiden Kliniken in die Helios Kliniken GmbH the area of neurology is currently being expanded. Together integriert. Die Beziehungen des MDC zu dem Klinikum Buch with its partner institute, the Institute for Molecular Pharma- im neurobiologischen Bereich werden im Moment ausgebaut. cology (FMP) and the approximately 30 firms on the Buch Zusammen mit dem Partner-Institut des MDC, dem For- Campus (in part new businesses founded by the MDC and the schungsinstitut für Molekulare Pharmakologie (FMP), und research clinics), the MDC has virtually the entire range of den rund 30 Firmen auf dem Bucher Campus, die zum Teil research and clinical tools for molecular medicine at its Ausgründungen des MDC und der Forschungskliniken sind, disposal. In addition to advances in genetics and cell biology, verfügt das MDC somit über nahezu die ganze Bandbreite an MDC scientists are able to analyze the structures of essential Forschungswerkzeugen, um Krankheiten molekular zu cha- macromolecules and to develop interacting substances for rakterisieren. Neben der genetisch/zellbiologischen Beschrei- potential therapeutic purposes. Such substances can then be bung der Krankheiten können die Bucher Wissenschaft- tested in pre-clinical and clinical studies on the Buch Campus lerinnen und Wissenschaftler die Struktur von essentiellen and, in some cases, engineered for use as clinical therapies. Makromolekülen analysieren und Substanzen entwickeln, die mit ihnen in Wechselwirkung treten können, um sie zum 9

Beispiel zu blockieren. Diese Substanzen können in vorklini- schen und klinischen Studien getestet und verbessert werden und, so ist die Hoffnung, potentiell in der Diagnostik und Therapie von Patienten eingesetzt werden.

Sie sehen drei Teile im Wissenschaftlichen Bericht, die sich mit der Forschung in diesen drei Bereichen – Herz-Kreislauf- forschung, Krebsforschung und Neurowissenschaften – be- schäftigt. Sie sind in Englisch geschrieben und für Wissen- schaftler und Studenten, auch potentielle neue Mitarbeiter, gedacht. Essentielle Teile des Berichtes sind in Deutsch ver- fasst, um diesen Bericht auch der breiten Öffentlichkeit des MDC zugänglich zu machen. Ein zusätzlicher Teil beschäf- tigt sich mit neuen Entwicklungen in der Verwaltung und den technischen Abteilungen des MDC.

Wichtig war in der Berichtsperiode, dass wir erfolgreich wichtige Bleibeverhandlungen mit leitenden Wissenschaft- lern des MDC führen konnten, die auswärtige Rufe erhalten hatten. Mehrere weitere Berufungen von auswärtigen, international anerkannten Forschern sind im Moment im Gange. Die drei Forschungsbereiche des MDC sind in der Berichts- periode von der Helmholtz-Gemeinschaft im Rahmen der programmorientierten Forschung (PROF) international begutachtet worden, und sie haben exzellent abgeschnitten. Zukünftig werden zwei strategische Ziele von übergeord- netem Interesse für das Institut sein. Zum einen wird die Kli- nische Forschung stärker mit der grundlagenorientierten „Junger Mann mit Schal“ (Portrait Max Delbrück) um 1935–40, Jeanne Mammen Forschung vernetzt werden. Zum zweiten werden die For- Copyright: Archiv Jeanne-Mammen-Gesellschaft e. V., Photograph: Gunter Lepkowski “Young Man with Shawl” (Portrait of Max Delbrück) about 1935-40, Jeanne Mammen schungsprogramme die Zusammenarbeit mit den univer- Copyright: Archiv Jeanne-Mammen-Gesellschaft e.V., Photographer: Gunter Lepkowski sitären Einrichtungen weiter ausbauen. Im Hinblick auf diese Ziele wird das MDC gemeinsam mit der Charité Univer- sitätsmedizin Berlin ein neues Experimental and Clinical This Research Report contains three parts which correspond Research Center (ECRC) auf dem Campus Berlin-Buch to the three main research areas: cardiovascular diseases, errichten. cancer, and neuroscience. The research sections are intended for scientists and students, but also for potential new MDC Wir wünschen Ihnen beim Studium dieses Forschungsberich- collaborators. Organizational sections of the Research Report tes viel Vergnügen. have been written in English and in German in order to make them accessible to the general public. Detlev Ganten Walter Birchmeier

During the period of the Report, we were able to successfully carry out essential negotiations with important senior scientists at the MDC who had received offers from national and foreign universities and research institutes. Several MDC appointments of internationally recognized researchers are currently in process. In addition, during the 2002-2003 period, the three research areas of the MDC were evaluated by international experts through the Helmholtz Association (HGF) within the framework of the program-oriented research (PROF). The MDC received an excellent overall rating. Two strategic objectives are of future interest for the Institute. First, clinical research will be more intensively linked with basic research. Secondly, the collaborations between the MDC research programs and the university institutions will increase. To meet these objectives, the MDC is establishing the Experimental and Clinical Re- search Center (ECRC) on the Berlin-Buch Campus in cooperation with the Charité University Medical School Berlin.

We hope you enjoy reading the MDC Research Report 2004.

Detlev Ganten Walter Birchmeier 10 Cardiovascular and Metabolic Diseases

Hypertension, Vascular Disease, and Kidney Disease Coordinator: Thomas Willnow

Heart Disease Coordinator: Ludwig Thierfelder

Metabolic Diseases, Genetics, Genomics, and Bioinformatics Coordinator: Jens Reich 12

Cardiovascular and Metabolic Herz-Kreislauf- und Diseases Stoffwechselerkrankungen

Thomas E. Willnow Thomas E. Willnow

Introduction Einführung

Diseases of the cardiovasculature and the metabolism are the Erkrankungen des kardiovaskulären Systems und des Stoff- major cause of morbidity and mortality in our society. wechsels sind die Hauptursache von Morbidität und Morta- Because such disorders particularly affect the elderly, the lität in unserer Gesellschaft. Auf Grund eines deutlichen socio-economic impact of these disease entities is expected to Anstiegs der durchschnittlichen Lebenserwartung in unserer rise even further in aging populations of the Western world. Bevölkerung und des erhöhten Risikos älterer Mensch an Research in this program aims at elucidating the genes and kardiovaskulären Komplikationen zu erkranken, ist davon genetic pathways that regulate the normal function of the auszugehen, dass die Belastungen unserer Gesundheits- cardiovascular system and the metabolism as well as those systeme durch die Folgekosten kardiovaskulärer Krankheiten that cause human disease in these areas. Ultimately, identi- zukünftig dramatisch ansteigen werden. Ziel unserer For- fication of disease genes will lead to a better understanding of schungsanstrengungen ist es, die genetischen Mechanismen cardiovascular disease processes, to improved diagnosis, and aufzuklären, welche die normalen Funktionen von Herz- to new concepts in therapy. Toward achieving these goals, we Kreislauf und Stoffwechsel regeln und welche für krankhafte use functional genomics approaches to study disease pro- Veränderungen dieser Systeme beim Patienten verantwortlich cesses in many systems that provide utilitarian models sind. Letztlich wird die Identifizierung grundlegender gene- including fruit fly, zebrafish, frog, mouse, rat, and compare tischer Mechanismen zu einem besseren Verständnis kardio- our findings to studies conducted in human (and vice versa). vaskulärer Krankheitsprozesse, zu verbesserter Diagnostik Our studies are performed by scientists that lead research und zu neuen therapeutischen Ansätzen führen. Um dieses groups at the MDC in close collaboration with clinicians at Ziel zu erreichen, verfolgen wir ein Konzept der vergleichen- the Franz-Volhard-Clinic for Cardiovascular Diseases (FVK). den Genomforschung, bei dem wir normale physiologische These research activities are coordinated in three topics that Prozesse und krankhafte Veränderungen des kardiovas- are of particular relevance to this program, namely: kulären Systems parallel in Patienten und in experimentellen Tiermodellen untersuchen und mit einander vergleichen. Aus (1) Hypertension, Vascular and Kidney Diseases den Informationen, welche wir in Modellsystemen wie der (2) Heart Diseases Fruchtfliege, dem Krallenfrosch oder Nagern gewinnen, (3) Genetics, Genomics, Bioinformatics, and Metabolism. lassen sich wichtige Rückschlüsse auf relevante Krankheits- prozesse beim Menschen ziehen und neue Strategien zu deren Therapie entwickeln. Unsere Arbeiten sind das Ergebnis einer engen Kooperation von Grundlagenwissenschaft am MDC und klinischer Forschung an der Franz-Volhard-Klinik für Herz-Kreislauferkrankungen (FVK) der Charité-Univer- sitätsmedizin Berlin auf dem Campus Berlin-Buch. Unsere Forschungsaktivitäten konzentrieren sich auf drei Themen- felder mit besonders hoher Relevanz für kardiovaskuläre Erkrankungen:

(1) Hypertonie, Gefäß- und Nierenerkrankungen (2) Herzerkrankungen (3) Genetik, Genomik, Bioinformatik und Metabolismus. 13

Hypertension, Vascular, and Kidney Diseases Hypertonie, Gefäß- und Nierenerkrankungen

Hypertension is a complex regulatory disorder that results in Hypertonie, die krankhafte Erhöhung des Blutdrucks, ist eine increased blood pressure. The heart, the blood vessels, and komplexe Regulationsstörung des Kreislaufs, welche in unse- the kidney are involved either as a primary cause or as a rer Bevölkerung weit verbreitet ist. Fehlfunktionen des Her- secondary target of this disease. With the elucidation of zens, der Gefäße oder der Nieren sind primäre Ursache dieser hitherto unknown genetic mechanisms contributing to hyper- Störung oder treten sekundär als Folge pathologischer Verän- tension, vascular, and kidney disease, new therapies may derungen beim Hypertoniker auf. Durch die Entschlüsselung become possible. Major scientific achievements in this pro- bislang unbekannter genetischer Mechanismen, die zu Blut- gram topic in the last two years include the identification of hochdruck, zu Gefäßerkrankungen oder zu Nierendefekten novel signaling pathways in platelets involving serotony- führen, hoffen wir die Ursachen der Hypertonie aufzuklären lation of small GTPases by the group of Michael Bader. In und neue Strategien zu deren Prävention entwickeln zu kön- addition, Friedrich Luft and colleagues were able to fine map nen. Zu unseren bedeutensten wissenschaftlichen Leistungen a gene locus on chromosome 12p responsible for autosomal- der vergangenen 2 Jahre zählen die Arbeiten der Gruppe um dominant hypertension, while Thomas Willnow and co- Michael Bader über die zentrale Rolle serotonin-vermittelter workers uncovered the molecular mechanism responsible for Prozesse bei der Blutplättchenaggregation und der Throm- the renal uptake of aminoglycoside antibiotics paving the bose-Entstehung. Friedrich Luft und Kollegen gelang es, way for future strategies to prevent nephrotoxic side effects einen Genort auf Chromosom 12 zu kartieren, der eine erb- of these widely used therapeutics. liche, autosomal-dominante Form der Hypertonie beim Menschen bedingt. Der Verfasser dieses Beitrags (Thomas Willnow) und seine Mitarbeiter konnten den Mechanismus Heart Diseases aufdecken, der für die Nephrotoxizität von Aminoglykosiden verantwortlich ist, eine lebensbedrohliche Nebenwirkung der Coronary artery disease, myocardial infarction, heart failure, Therapie mit diesen Antibiotika. and cardiomyopathies are the main research areas in this topic. Primarly, we focus on the identification of disease genes that underlie monogenic traits in patients and in animal Herzerkrankungen models as an approach to understand the molecular basis of heart disease. Major achievements in recent years include the Erkrankungen der Koronargefäße, Herzinfarkt, Herzversagen identification of mutations in the muscle protein titin as a und Kardiomyopathien sind die Hauptforschungsfelder die- cause of hereditary cardiomyopathy in affected families ses Themas. Wir konzentrieren uns dabei vorrangig auf die (Luwdig Thierfelder and colleagues) and the pathophy- Kartierung neuer Krankheitsgene, welche Ursache erblicher siological characterization of this protein in a mouse model Herzerkrankungen beim Menschen sind. Begleitend hierzu of titin deficiency (Michael Gotthardt). Salim Abdelilah- erstellen wir transgene Tiermodelle, welche dem Patienten Seyfried’s laboratory characterized the crucial role of atypi- vergleichbare genetische Veränderungen tragen und uns eine cal protein kinase C in determination of epithelial cell detaillierte Untersuchung pathologischer Vorgänge ermög- polarity and heart development, while Gerd Wallukat charac- lichen. Zu unseren wesentlichen Ergebnissen der letzten terized autoantibodies against G-protein coupled receptors as Jahre zählen der Nachweis von Mutationen im Muskelprotein a mechanism contributing to myocarditis, dilated cardio- Titin als Ursache einer erblichen Form der Kardiomyopathie myopathy, and hypertension in patients. beim Patienten (Ludwig Thierfelder und Kollegen) sowie die pathophysiologische Charakterisierung dieses Proteins in einem Mausmodell mit induziertem Titin-Gendefekt (Michael Genetics, Genomics, Bioinformatics, and Gotthardt). Das Labor von Salim Abdelilah-Seyfried konnte Metabolism die zentrale Rolle bearbeiten, welche die atypische Protein- kinase C bei der Regulation der Epithelzell-Polarität und der Elucidation of the human and other mammalian genomes Herzentwicklung spielt. Gerd Wallukat hat Autoantikörper heralds a new area in biomedical research. Major challenges gegen G-Protein-gekoppelte Rezeptoren als Ursache dilata- in the future will be to assign functions to the wealth of tiver Kardiomyopathien identifiziert. sequence information generated in the various genome projects. Thus, high throughput sequence analysis and bioinformatics technologies have to be developed and applied to Genetik, Genomik, Bioinformatik und Metabolismus the positional cloning of disease genes in monogenic and complex traits. Toward these goals, recent major achieve- Die Entschlüsselung des Erbguts des Menschen und das ments in this program have been the positional cloning of anderer Säuger ist ein Meilenstein der modernen biomedizi- disease genes responsible for autosomal-recessive hyper- nischen Forschung. Noch größer jedoch als die Herausforde- cholesterolemia (Friedrich Luft), familial hypertrophic rung der Entschlüsselung des menschlichen Erbguts ist die cardiomyopathy (Peter Nürnberg and Karl-Josef Osterziel), Aufgabe, die gewonnene genetische Information auszuwer- as well as nephronophthisis, and related nephritic disorders ten und funktionell zu charakterisieren. Ein wesentliches Ziel (Peter Nürnberg). des Forschungsschwerpunktes Genetik, Genomik und Bio- informatik besteht darin neue molekulargenetische, bioinfor- matische und biostatistische Verfahren zu entwickeln, um die Flut genetischer Information zu analysieren und für funktio- 14

nelle Untersuchungen wie der Kartierung von Krankheitsge- nen einzusetzen. Unsere Arbeiten der letzten Jahre haben sich im Wesentlichen auf die Anwendung dieser Technologien zur Hochdurchsatz-Analyse monogenetischer und komplexer genetischer Krankheiten fokussiert. Wichtigste Ergebnisse dieser Arbeiten waren die positionelle Klonierung der Gene für autosomal-rezessive Hypercholesterinämie (Friedrich Luft), für familiäre hypertrophe Kardiomyopathie (Peter Nürnberg und Karl-Josef Osterziel) sowie für Nephro- nophthise (Schrumpfniere) und verwandte nephritische Störungen (Peter Nürnberg). Hypertension, Vascular Disease, and Kidney Disease

Molecular Cardiovascular Research circumvent the problem of perinatal lethality. Using mice with kidney-specific megalin deficiency, we identified megalin as a receptor for vitamin D binding protein (DBP), the

Thomas E. Willnow plasma carrier for the steroid 25-(OH) vitamin D3, and demonstrated that the receptor mediates the tubular retrieval

of 25-(OH) vitamin D3-DBP-complexes filtered through the glomerulus. This receptor-mediated uptake is required to

prevent the loss of vitamin D3 metabolites by glomerular filtration. Furthermore, it delivers 25-(OH) vitamin D3 to tubular epithelial cells for conversion into 1, 25-(OH)2 vitamin D3, the active form of the vitamin and a potent regulator Introduction of the systemic calcium and bone metabolism. Urinary excre-

tion of 25-(OH) vitamin D3 in megalin-deficient mice results The low-density lipoprotein (LDL) receptor is a 150-kDa in vitamin D deficiency and in impaired bone calcification. endocytic receptor that mediates the cellular uptake of lipo- Thus, megalin acts as an endocytic receptor for the uptake of protein particles and plays a central role in the removal of lipophilic vitamin D and controls a central regulatory step in lipids from the systemic circulation. In patients with a genetic bone metabolism. Ongoing research is directed towards the defect of the LDL receptor (Familial Hypercholesterolemia), elucidation of the role of megalin in forebrain development massive increase in the concentration of circulating plasma and in holoprosenencephaly, a defect that may be caused by lipoproteins results in hyperlipidemia and results in athero- impaired embryonic cholesterol metabolism and that is among sclerosis and coronary artery disease. In recent years, a the most common developmental defects of the human number of novel receptors have been identified that are struc- embryo (1 in 250 pregnancies). turally related to the LDL receptor and are designated members of the LDL receptor gene family (figure 1). Given the central role of the LDL receptor in the cardiovascular Role of megalin in aminoglycoside-induced system, equally important roles for other receptors in this nephrotoxicity gene family are anticipated. The focus of our studies is the elucidation of the functions that receptors of the LDL recep- Besides clearance of endogenous ligands from the primary tor gene family play in the (patho)physiology of the lipid urine, megalin is also responsible for retrieval of foreign metabolism. Towards this goal, we are using gene targeting substances filtered through the glomerulus. This activity is approaches to generate mouse models with deficiencies in particularly relevant for the renal uptake of therapeutic drugs LDL receptor-related receptors and to analyze the consequen- that accumulate in the kidney causing nephrotoxicity. Such ces of the receptor gene defects in vivo. In recent studies, we drugs include aminoglycosides, antibiotics commonly used to have identified important new functions of lipoprotein recep- treat life-threatening gram negative bacterial infections. tors in bone metabolism, brain development and male fertility Aminoglycosides were known to accumulate in the renal as well as the molecular mechanisms underlying human proximal tubules causing nephrotoxicity and kidney failure. diseases in these areas. However, the pathway responsible for renal uptake of the antibiotic remained elusive. We have used mouse models with genetic megalin deficiency to explore the contribution of this Role of megalin in renal uptake of steroid hormones receptor to renal aminoglycoside uptake in vivo. We demonstrated that the uptake of aminoglycosides into the kidney We have focused on the functional characterization of mega- directly correlates with renal megalin activity and is comple- lin, a member of the LDL receptor gene family predominantly tely eliminated in mice lacking the receptor. Thus, our studies expressed in the neuroepithelium of the developing embryo provide unequivocal genetic evidence that megalin is the and in proximal tubules of the adult kidney. Targeted disrup- major pathway responsible for renal aminoglycoside accumu- tion of the respective gene in the mouse results in develop- lation and that the receptor represents a unique drug target to mental defects of the forebrain (holoprosencephaly) and in prevent aminoglycoside-induced nephrotoxicity. In colla- perinatal lethality of affected animals. Because we were boration with pharmaceutical partners, we are currently particularly interested in elucidating the role of the receptor in developing antagonists that block aminoglycoside binding to the adult kidney, we used conditional gene targeting to gene- megalin and that may be used in the future to prevent amino- rate mice with a kidney-specific megalin gene defect and to glycoside-induced kidney damage in patients. 16

Figure 1) Structural organization of selected members of the LDL receptor gene family and related Vps10p receptors. Structural elements found in the various receptor species are depicted. These elements include ligand binding type (filled dots) and epidermal growth factor (EGF) precursor type repeats (open dots), transmembrane domains (filled square), fibronectin type III repeats (open square) and Vps10p homology domain (filled oval).

Role of the apolipoprotein receptor E-2 in sperm Functional characterization of cellular sorting development receptors

The apolipoprotein (apo)E receptor-2 (apoER2) is another Previously identified members of the LDL receptor gene member of the low-density lipoprotein receptor gene family family exhibit structural motifs found in the LDL receptor and an important regulator of neuronal migration. Besides (figure 1). This observation suggests a role of these receptors acting as a lipoprotein receptor, the protein also functions as a in endocytosis of extracellular ligands, a hypothesis support- cellular receptor for the signaling factor Reelin and provides ed by our findings in receptor-deficient mouse models. positional cues to neurons that migrate to their proper Recently, a novel receptor sorLA-1 was uncovered that position in the developing brain. Loss of receptor activity in a combines motifs of the LDL receptor gene family with struc- knockout mouse model results in false layering of neurons in tural elements found in the yeast vacuolar sorting receptor the central nervous system. Besides brain defects, apoER2- Vps10p (figure 1). SorLA-1 in turn is highly homologous to a deficient mice also exhibit male infertility suggesting a as of novel class of mammalian Vps10p-related receptor (sortilins, yet unknown role of the receptor in male reproduction. We sorCS) (figure 1). Although, several of these receptors have demonstrated that apoER2 is highly expressed in the initial been shown to bind ligands relevant for lipoprotein meta- segment of the epididymis where it affects the functional bolism such as apolipoprotein E or lipoprotein lipase, the expression of phospholipid hydroperoxide glutathione physiological role of these sorting receptors and their rele- peroxidase (PHGPx), an enzyme required for sperm matura- vance in cellular and systemic lipid metabolism is unclear at tion. Reduced PHGPx expression in apoER2 knockout mice present. Here, we have generated knockout mouse models results in the inability of the sperm to regulate the cell volume lacking functional expression of the various members of this and in abnormal sperm morphology and sperm immotility gene family. Using these models, we have uncovered the fun- (figure 2). Because insufficient expression of PHGPx is a ction of sortilin as a novel receptor for nerve growth factor in major cause of infertility in men, these findings not only high- the peripheral nervous system. Nerve growth factor (NGF) light an important new function for apoER2 that is unrelated regulates neuronal development through both survival and to neuronal migration but also suggest a possible role for death signalling via two distinct receptors, TrkA and p75NTR. lipoprotein receptors in human infertility. Ongoing research Both NGF and its precursor proNGF are released by cells, but activities are aimed at unraveling the molecular mechanisms in contrast to NGF that induces cell survival, proNGF selec- causing male infertility in these conditions. tively promotes p75NTR-mediated apoptosis. We demonstrated that Sortilin, a receptor expressed in proNGF-responsive tissues, is indispensable for the pro-apoptotic effect of proNGF. Sortilin binds to the pro-domain of proNGF whereas p75NTR selectively binds to the mature NGF domain. Together, both receptors form a composite receptor binding 17

osteopathy in mice with kidney-specific megalin gene defect. FASEB J. 17: 247-249.

Andersen, O.M., Yeung, C.-H., Vorum, H., Wellner, M., Andreassen, T.K., Erdmann, E., Mueller, E.-C., Herz, J., Otto, A., Cooper, T.G. and T. E. Willnow. (2003) Essential role of the Apolipoprotein E receptor-2 in sperm development. J. Biol. Chem. 278:23989-23996.

Schmitz, C., Hilpert, J., Boensch, C., Christensen, E.I., Luft, F.C. and T.E. Willnow. (2002) Megalin deficiency protects from renal aminoglycoside accumulation. J. Biol. Chem. 277: 618-622.

Nykjaer, A. and T.E. Willnow. (2002) The LDL receptor gene family: A Cellular Swiss army knife? Trends Cell Biol. 12: 273-280.

Structure of the Group

Group Leader Prof. Dr. Thomas E. Willnow

Scientists Dr. Olav Andersen Dr. Tilman Breiderhoff* Dr. Annette Hammes-Lewin* Dr. Helle Heibroch Petersen

Graduate Students Uwe Anzenberger* Regina Burmeister* Robert Spoelgen Juliane Reiche

Technical Assistants Figure 2) Abnormal sperm tail morphology in apolipoprotein receptor E-2 Marc Eigen deficient mice. Sperm of apoEr-2 deficient mice (-/-) are characterized by abnormal bending of the sperm tail (arrows) causing sperm immotility and male infertility. Sperm Charlotte Räder of wild type mice (+/+) with normal straight tail morphology are shown for comparison. Hannelore Schulz Donate Vetter Susanne Schütz*

Secretariat Verona Kuhle site on target cells responsible for mediating the pro-apoptotic effect of proNGF. Ongoing studies are aimed at identifying * part of the period reported the role of the neurotrophin receptor Sortilin and related Vps10p receptors in the cardiovasculature.

Selected Publications

Nykjaer, A., Lee, R., Teng, T.T., Nielsen, M.S., Jansen, P., Madsen, P., Jacobsen, C., Kliemannel, M., Willnow, T.E., Hempstead, B. and C.M. Petersen (2004). A novel neurotrophin receptor essential for proNGF induced neuronal cell death. Nature 427: 843-848.

Leheste, J., Melsen, F., Wellner M., Jansen, P., Schlichting U., Renner-Müller I., Andreassen T.K., Wolf, E., Bachmann, S., Nykjaer A. and T. E. Willnow. (2003) Hypocalcemia and 18

Molecular Biology and Genetics of Transgenic technology Cardiovascular Diseases In order to study the functional relevance of genes linked to hypertension and stroke, transgenic rats are produced with Detlev Ganten alterations in the expression of these genes. The power of this technology has been demonstrated in several transgenic rat models with modifications in the renin-angiotensin system. Rats expressing the mouse renin-2 gene (TGR(mREN2)27) have helped to reveal the physiological functions of local renin-angiotensin systems in tissues, in particular in the central nervous system.

It was shown that overactivation of the renin-angiotensin system in the brain markedly contributes to the pathogenesis of hypertension in this model. Furthermore, double transgenic rats carrying the human renin and angiotensinogen genes have been generated and turned out to be excellent models to study pregnancy-induced hypertension and hypertension- induced end-organ damage in kidney and heart. These rats are also used as suitable models for the development of renin inhibitors which represent a novel therapeutic approach to inhibit the renin-angiotensin system in hypertension and other Mapping hypertension genes cardiovascular diseases. In addition, numerous other transgenic rat models for the study of cardiovascular physiology The rat is one of the most important model systems for com- and of the pathophysiology of cardiovascular and other dis- plex, polygenic diseases. We have demonstrated that multiple eases, such as Alzheimer’s and Huntington’s, have been gene- chromosomal loci in rat models contribute to blood pressure rated and analyzed in collaboration with other groups. regulation and hypertension. Independent from elevated blood pressure, additional genetic factors contribute to end- Transgenic technology in the rat is developed further by the organ damage and stroke in these animals. Ongoing research optimization of the methodology, the generation of transgenic in our laboratory is directed towards the identification of the animals with large genomic constructs, the use of additional underlying predisposing genes and the subsequent identifi- strains of rats, and first steps toward the establishment of cation of their molecular variants which cause different knockout technology for this species. It could be shown that cardiovascular disease phenotypes. To localize disease genes superovulation of Sprague-Dawley rats can be achieved as within chromosomal regions linked to quantitative traits (e.g. efficiently by single injections of pregnant mare’s serum blood pressure), we are establishing multiple congenic rat gonadotropin as by minipump infusions of follicle stimulat- strains by introgressing disease alleles encompassing the ing hormone. Furthermore, the efficiency of transgenic rat quantitative trait locus (QTL) into a non-affected reference production was shown to be independent of the transgene strain by successive backcrossing and molecular analysis. construct and overnight embryo culture did not diminish the This strategy allows the observation of the effect and the success rate of the method. In addition to the most frequently genetic analysis of a single QTL. We are currently applying used Sprague-Dawley strain, transgenic rats have been this strategy to a number of QTLs for blood pressure regulation, stroke, and kidney disease in the stroke-prone spontaneously hypertensive rat. A similar strategy is currently being adopted in collaboration with our Israeli partners to elucidate the genetic basis of salt-sensitive hypertension in Rat oocytes after somatic nuclear transfer the Sabra rat model. The combination of congenic experimentation with the development of subcongenic animals, with only a fraction of the initial congenic segment, will facilitate successive fine mapping within a QTL.

Analysis of the rat genome

As a partner in national and international rat genome research, our group has participated in the development of a large number of genomic resources for the rat to facilitate functional genomic studies in this species. These efforts are part of an international group of investigators led by the Baylor College of Medicine (Rat Sequencing Project Consor- tium) which have led to the sequencing and annotation of the rat genome. 19

successfully generated from Wistar-Kyoto (WKY), Lewis, Structure of the Group and spontaneously hypertensive rats (SHR-SP). Group Leader For the purpose of enabling knockout technology in the rat, Prof. Dr. Detlev Ganten the group has established embryonic stem (ES) cells from this species and has performed nuclear transfer experiments to Scientists allow the cloning of rats. Rat ES cells were developed from Dr. Norbert Hübner several strains including Sprague-Dawley and WKY but, de- Claudia Gösele spite expressing characteristic ES cell markers, these cells Dr. Maolian Gong were not able to participate in the development of a rat embryo and, thus, could not be used to generate chimeras and, Graduate Student finally, knockout rats. However, the cells were shown to Yinyan Sun promote acceptance of transplanted organs when injected into host animals one week prior to transplantation, opening up a Technical Assistants new therapeutic option using ES cells in transplantation Heide Kistel medicine. Anita Müller

In order to enable the cloning of rats, an efficient culture system for rat preimplantation embryos was developed and enucleation and activation protocols for rat oocytes were optimized. First transfers of somatic cell nuclei into enuclea- ted rat oocytes have produced embryos which, however, could only be cultured for a limited period of time. Neverthe- less, the newly developed methods allowed the generation of tetraploid as well as parthenogenetic rat embryos as well as serve as a basis for the establishment of efficient nuclear transfer technology for the rat.

Selected Publications

International Rat Sequencing Project Consortium. (2004) Genome Sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428:493-521.

Aileru AA, Logan E, Callahan M, Ferrario CM, Ganten D, Diz DI. (2004) Alterations in sympathetic ganglionic transmission in response to angiotensin II in (mRen2)27 transgenic rats. Hypertension. 43:270-275.

Krivokharchenko A, Popova E, Zaitseva I, Vilianovich L, Ganten D, Bader M. (2003) Development of parthenogenetic rat embryos. Biol Reprod. 68:829-836.

Finckenberg P, Inkinen K, Ahonen J, Merasto S, Louhelainen M, Vapaatalo H, Müller D, Ganten D, Luft F, Mervaala E. (2003) Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways. Am J Pathol. 163:355-366.

Fändrich F, Lin X, Chai GX, Schulze M, Ganten D, Bader M, Holle J, Huang DS, Parwaresch R, Zavazava N, Binas B. (2002) Preimplantation-stage stem cells induce long-term allogeneic graft acceptance without supplementary host conditioning. Nat Med. 8:171-178. 20

Medical Genomics These data are being used to identify clusters of genes that co- segregate with well-documented cardiovascular and metabolic phenotypes within spontaneously hypertensive rats and Norbert Hübner to identify the underlying allelic variants. By determining the genetic networks and regulatory mechanisms underlying the observed patterns of gene expression, these data will provide new insights into the control mechanisms for hypertension, insulin resistance, and associated metabolic phenotypes that may be shared in common with similar disorders in humans.

The identification of disease-relevant genes within QTLs by positional cloning will be greatly facilitated once the sequence of the entire rat genome is known. Moreover, functional studies often require access to clones in specific regions of interest. We have thus participated in the Rat Genome Sequencing Project Consortium, which resulted in the identification and annotation of the entire rat genome sequence. Additionally, we have built a physical map based on Yeast Artifical Chromosomes (YAC) and Bacterial Arti- fical Chromosomes (BAC). Combined this map comprises more then 200,000 BAC and YAC clones which are all anchored to the genomic sequence. These clones provide Analysis of complex cardiovascular disorders ready access to any genomic region for functional studies. in the rat

The rat is one of the most important model systems for Selected Publications complex, polygenic diseases. Since all epidemiologically important human diseases belong to this category, the potential International Rat Sequencing Project Consortium. (2004) for major advances through genetic investigation is substantial. Genome Sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428: 493-521. We have demonstrated that multiple chromosomal loci in rat models contribute to blood pressure regulation and hyper- Martin Krzywinski, John Wallis, Claudia Gösele, Ian Bosdet, tension. Independent from elevated blood pressure, additional Readman Chiu, Tina Graves, Oliver Hummel, Dan Layman, genetic factors contribute to end-organ damage and stroke in Carrie Mathewson, Natasja Wye, Baoli Zhu, Derek Albracht, these animals. Ongoing research in our laboratory is directed Jennifer Asano, Sarah Barber, Mabel Brown-John, Susanna towards the identification of the underlying predisposing Chan, Steve Chand, Alison Cloutier, Jonathon Davito, Chris genes and the subsequent identification of their molecular Fjell, Tony Gaige, Detlev Ganten, Noreen Girn, Heinz variants, which cause cardiovascular disorders. Himmelbauer, Thomas Kreitler, Stephen Leach, Darlene Lee, Hans Lehrach, Michael Mayo, Kelly Mead, Teika Olson, To localize disease genes within chromosomal regions linked Pawan Pandoh, Anna-Liisa Prabhu, Heesun Shin, Miranda to quantitative traits (e.g. blood pressure), we are establishing Tsai, Jason Walker, George Yang, Mandeep Sekhon, LaDeana multiple congenic rat strains by introgressing disease alleles Hillier, Heike Zimdahl, Simone Tänzer, Kazutoyo Osoegawa, encompassing the quantitative trait locus (QTL) into a non- Shaying Zhao, Asim Siddiqui, Pieter J. de Jong, Wes Warren, affected reference strain by successive backcrossing and Elaine Mardis, John D. McPherson, Rick Wilson, Norbert molecular analysis. This strategy allows the observation of Hübner, Steven Jones, Marco Marra, Jacqueline Schein. the effect and the genetic analysis of a single QTL. We are (2004) Integrated and sequence-ordered BAC and YAC-based currently applying this strategy to a number of QTLs for physical maps for the rat genome. Genome Research (in blood pressure regulation, stroke, and kidney disease in the press). stroke-prone spontaneously hypertensive rat. Heike Zimdahl, Gerald Nyakatura, Petra Brandt, Herbert The combination of congenic experimentation with the deve- Schulz, Oliver Hummel, Berthold Fartmann, David Brett, lopment of subcongenic animals, with only a fraction of the Marcus Droege, Jan Monti, Young-Ae Lee, Yinyan Sun, initial congenic segment will enable successive fine mapping Shaying Zhao, Ethan E. Winter, Chris P. Ponting, Yuan Chen, within a QTL. The mapping efforts of complex cardiovascular Arek Kasprzyk, Ewan Birney, Detlev Ganten, Norbert traits by congenic experimentation and positional cloning will Hübner. (2004) A SNP map of the rat genome generated from be used in ongoing projects jointly with the establishment of cDNA sequences. Science 303: 807. gene expression signatures in target organs of congenic animals and their parental progenitors. High density microarrays are used for this approach. A combinatorial approach of positional cloning and expression profiling will provide a powerful tool to identify positional candidate genes within chromosomal regions for genetically determined cardiovascular diseases. 21

Graduate and Undergraduate Students Judith Fischer Lalitha Kato Yinyan Sun

Technical Assistants Susanne Blachut Heide Kistel Anita Müller Sabine Schmidt

Anchoring of fingerprint and YAC map regions to sequence assembly. YAC contigs are anchored to the assembly by way of hybridizations to BACs with sequence coordinates. The sequence coverage of anchoring BACs is typically less than the estimated size of the YAC contigs and therefore the contigs as drawn do not reflect actual size. Light grey lines link regions that are (a) are anchored by hybridization to a single BAC that is associated with <80% of the contig YACs or (b) are anchored by <20% of the contig BACs, leading to spurios contig segmentation on the assembly not likely to be dueto actual inconsistencies between the YAC map and the sequence assemly. Red lines link the remaining region pairs, for which segmentation evidence is robust.

Maolian Gong, Hongye Zhang, Herbert Schulz, Young-Ae Lee, Kai Sun, Sylvia Bähring, Friedrich C. Luft, Peter Nürnberg, Andre Reis, Klaus Rohde, Detlev Ganten, Rutai Hui, Norbert Hübner. (2003) Genome-wide linkage reveals a locus for human essential (primary) hypertension on chromosome 12p. Human Molecular Genetics 12:1273-1277.

Jan Monti, Ralph Plehm, Herbert Schulz, Detlev Ganten, Reinhold Kreutz, Norbert Hübner. (2003) Interaction between blood pressure quantitative trait loci in rats in which trait variation at chromosome 1 is conditional upon a specific allele at chromosome 10. Human Molecular Genetics 12: 435-439.

Structure of the Group

Group Leader Dr. Norbert Hübner

Scientists Claudia Gösele Dr. Maolian Gong Oliver Hummel Dr. Jan Monti Dr. Herbert Schulz Dr. Yaxin Xu Dr. Heike Zimdahl 22

Molecular Biology KKS in an intact animal, transgenic rats were generated of Peptide Hormones expressing the human tissue kallikrein gene in all organs tested and excreting the protein in the urine. In these rats, blood pressure and its diurnal rhythmicity as measured by Michael Bader telemetry is significantly reduced compared to control rats. Moreover, kidneys and hearts of the animals are protected against ischemic and hypertrophic injury.

The functions of the kinin B1 receptor are enigmatic. There- fore, we generated mice lacking this subtype. The resulting animals exhibited analgesia, altered inflammatory reactions and reduced neovascularization, demonstrating an important role of the B1 receptor in pain transmission, inflammation, and angiogenesis. Recently, mice lacking both kinin receptors, B1 and B2, have been generated by deleting the B1 gene in embryonic stem (ES) cells derived from B2 knockout mice. These animals are totally unresponsive to kinins and show complete protection from septic-shock induced hypotension.

Serotonin system

The group is interested in the molecular biology and function Serotonin is at the same time a very important neurotrans- of hormone systems involved in cardiovascular regulation. mitter in the brain and a major factor released by platelets in Besides the cloning and characterization of genes for the the circulation. In order to functionally characterize this components of these systems, the physiological functions of hormone, we deleted the gene encoding the rate limiting the systems are analyzed by the production and analysis of enzyme for its synthesis, tryptophan hydroxylase (TPH), transgenic and gene-targeted animal models. from the mouse genome. The resulting mice were depleted from serotonin in the circulation but, surprisingly, showed normal serotonin levels in the brain. This led us to detect and Renin-angiotensin system characterize a second gene coding for TPH, TPH2, responsible for serotonin synthesis in the central nervous system. The renin-angiotensin system (RAS) is centrally involved in The TPH1 deficient mice exhibited defects in platelet funct- blood pressure regulation and, therefore, has been studied in ion due to a blunted release of von Willebrand factor (vWF) at detail employing transgenic techniques. A major focus of our sites of vessel injury. Further analysis revealed that serotony- research is local angiotensin-II generating systems in tissues lation of small GTPases is a novel and essential signalling such as brain, heart, and kidney. Transgenic rats expressing an pathway in the release of platelet -granules. antisense-RNA against angiotensinogen exclusively in astrocytes of the brain were produced and showed a decreased local concentration of this protein and lowered blood pressure and plasma vasopressin levels. Using these rats, we could show that central angiotensin modulates circadian blood pressure rhythms and the baroreceptor reflex. Furthermore, it is involved in the hypertensive and hypertrophic effects of circulating angiotensin. In a transgenic mouse model lacking Serotonin in intestine and brain of normal (left panels, +/+) and TPH1-deficient (right panels, -/-) mice. Serotonin is absent from chromaffin cells of the gut (upper panels) of angiotensinogen synthesis in heart and kidney, a role of local TPH1-deficient mice but present in normal amounts in the Raphe nuclei of the angiotensin-II generation in these organs could be demonstra- brainstem (lower panels) due to the existence of a second tryptophan hydroxylase gene, TPH2, exclusively expressed in the brain. ted for the pathogenesis of hypertensive end-organ damage. Recently, new components of the RAS such as ACE2, the renin receptor, the mas protooncogene and angiotensin (1-7) have become a subject of research and transgenic animal models for the functional analysis of these molecules have been developed and characterized. Using knockout mice for the mas protooncogene, it could be shown that this protein is a functional receptor for angiotensin (1-7).

Kallikrein-kinin system

The kallikrein-kinin system (KKS) is an important hormone system for cardiovascular regulation mostly counteracting the effects of the RAS. As a model to study the functions of the 23

Transgenic and stem cell technology Structure of the Group

In collaboration with other groups, the expression of further Group Leader proteins with relevance for cardiovascular and other diseases PD Dr. Michael Bader have been altered by transgenic and knockout technology in mice and rats, such as the liver fatty acid binding protein, Scientists smooth muscle myosin heavy chain, natriuretic peptide recep- Dr. Natalia Alenina tors, and huntingtin. Dr. Ovidiu Baltatu Dr. Cibele Campos Cardoso In order to allow gene-targeting experiments also in the rat, Dr. Luciana Aparecida Campos which is more suitable for the research on cardiovascular Dr. Cécile Cayla* diseases than the mouse, we have established ES cells from Dr. Tanja Shmidt this species and performed nuclear transfer experiments to Dr. Radu Iliescu allow the cloning of rats. However, the rat ES cells did not Dr. Alexander Krivokharchenko form chimeras when injected into blastocysts and are there- Dr. Thomas Langenickel* fore not suitable for gene targeting experiments. Instead, they Dr. Elena Popova blunted transplant rejection when injected several days prior Dr. Mihail Todiras* to an allogenic heart transplanation. Dr. Diego Walther*

Graduate and Undergraduate Students Selected Publications Marcos Eduardo Barbosa* Saleh Bashammakh Walther, D.J., Peter, J.U., Bashammakh, S., Hörtnagl, H., Celine Burckle* Voits, M., Fink, H., and Bader, M. (2003) Synthesis of sero- Jens Buttgereit tonin by a second tryptophan hydroxylase isoform. Science Neil Docherty* 299, 76 Anderson Ferreira* Vanessa Ferreira Merino Walther, D.J., Peter, J.U., Winter, S., Höltje, M., Paulmann, Anna Panek* N., Grohmann, M., Vowinckel, J., Alamo-Bethencourt, V., Jens-Uwe Peter* Wilhelm, C.S., Ahnert-Hilger, G., and Bader, M. (2003) Sero- Brit Rentzsch* tonylation of small GTPases is a signal transduction pathway Larissa Vilianovitch that triggers platelet a-granule release. Cell, 115, 851-862 Claudia Wilhelm Ioulia Zaitseva Fändrich, F., Lin, X., Chai, G.X., Schulze, M., Ganten, D., Bader, M., Holle, J., Huang, D.S., Parvaresch, R., Zavazava, Technical Assistants N., and Binas, B. (2002) Preimplantation-stage stem cells in- Adelheid Böttger duce allogeneic graft tolerance without supplementary host Monika Nitz conditioning. Nat. Med. 8, 171-178 Liselotte Winkler Vera Saul* Morano, I.L., Chai, G.X., Baltas, L.G., Lamounier-Zepter, V., Lutsch, G., Kott, M., Haase, H., and Bader, M. (2000) Smooth Secretariat muscle contraction without smooth muscle myosin. Nat. Cell Dana Lafuente Biol. 2, 371-375. Manuela Friede-Strauch

Pesquero, J.B., Araujo, R.C., Heppenstall, P.A., Stucky, C.L., Silva, J.-A.Jr, Walther, T., Oliveira S.M., Pesquero, J.L., * part of the period reported Paiva, A.C., Calixto, J.B., Lewin, G.R., and Bader, M. (2000) Hypoalgesia and altered inflammatory responses in mice lacking kinin B1 receptors. Proc. Natl. Acad. Sci. USA 97, 8140-8145. 24

Genetics, Nephrology, the informative LDL/HDL ratio. Their informative haplo- Hypertension, and Vascular Injury types are currently being tested in other populations. Mono- zygotic and dizygotic twins continue to be a focus of the group. Andreas Busjahn has founded a company (Health- Friedrich C. Luft TwiSt GmbH) focusing on the twin model in elucidating genetic variance. However, the close association with the Nephrology/Hypertension section is being maintained.

Vasculitis

The group collaborated with the genetics group and relied on the twin model. They were able to show that ANCA expression on the neutrophil surface is highly influenced by genetic variance. Their findings raise the hope that genes regulating ANCA surface expression, and thereby susceptibility to the disease, can be isolated. Ralph Kettritz and Mira Choi were able to elucidate the functions of the nuclear factor Kappa-B (NF-B) in neutrophils by introducing a novel inhibitor (NEMO binding domain) into the cells. This feat was accomplished with help of the HIV protein TAT that is able to traverse cell membranes as a carrier of other molecules. Summary Recently, the group showed that beta2-integrins provide costimulatory signals for matrix proteins that are able to activate The group is interested in molecular genetics of cardiovascular NF-B in neutrophils. The signaling pathways involved are disease, pathogenesis of renal diseases, hypertension and vas- probably important in enabling neutrophils to tranverse cular injury. Sylvia Bähring leads a team focusing on genetics across capillary barriers in inflammatory conditions. of blood pressure regulation and lipid metabolism. Both Men- delian conditions and complex genetic diseases are being addressed. Ralph Kettritz is interested in small vessel disease, Vascular injury particularly anticytoplasmatic antibody-(ANCA) induced vasculitis. He is currently focusing on neutrophil biology. Domi- Dominik N. Müller and associates have convincingly demon- nik N. Müller is relying on a transgenic rat model to study strated that angiotensin (Ang) II sets a series of events in mechanisms of vascular injury. Volkmar Gross is pursuing motion that involve both innate and acquired immunity. The systems biology in the mouse. Various genetically modified immune reactions signal inflammatory events that result in animals are at his disposal. Anette Fiebeler is interested in vas- vascular injury. Dendritic cells are activated in this process, cular effects of aldosterone. Ralf Dechend is focusing on pre- grow to maturity, and migrate as a consequence of Ang II-in- eclampsia, a malignant form of hypertension during pregnancy. duced signaling. Blocking innate and acquired immunity, either pharmacologically or by depleting lymphocytes can ameliorate Ang II-induced vascular injury. In other studies, Molecular genetics the group is focusing on eicosanoid participation in Ang II-induced vascular injury (cooperation with Wolf-Hagen Sylvia Bähring and associates showed that autosomal-domi- Schunck). Aldosterone and the novel renin receptor have nant hypertension with brachydactyly is caused by a gene become recent focal points. Aldosterone signals via the mine- rearrangement on chromosome 12p (in press). The group ralocorticoid receptor in cardiac and smooth muscle cells. relied on interphase FISH and were joined by Anita Rauch The hormone augments Ang II-related effects. Reactive (University of Erlangen) in this endeavor. Rearrangements oxygen species and extracellular regulated kinase play a role have been identified in several families with this condition. in these responses. Mapping them precisely, cloning the breakpoints, and elucidating expressed sequence tags in the regions involved should enable the team to clone the responsible genes. Hussam Al- Systems biology in mice Kateb et al showed that autosomal-recessive hypercholesterolemia in a Syrian kindred is caused by a novel mutation in an Volkmar Gross and Michael Obst have elucidated Ang II LDL-receptor adaptor protein. He and his associates have receptor 2 (AT2) function with their work on AT2 receptor expressed the mutation in HEK cells and proved that the knockout mice. The AT2 receptor is important to shifts in pres- resulting mRNA was defective. Hans Knoblauch and associa- sure-natriuresis diuresis and also plays a role in the propensity tes performed a single nucleotide polymorphism (SNP) to develop cardiac hypertrophy. The group is able to measure screen in 13 lipid-relevant genes in a study of 250 German blood pressure and heart rate by telemetry and now have added families (>100 000 genotypes in >1000 people). They were continuous cardiac output measurements to their repertoire. able to explain most of the genetic variance in low-density They are currently focusing on soluble epoxide hypdrolase lipoprotein (LDL) cholesterol, high-density lipoprotein knockout mice and mice with a disrupted RGS2 gene. The gene (HDL) cholesterol, and almost all of the genetic variance in encodes a protein that regulates G-protein signaling. 25

by a TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis. Blood 102, 2259-67.

Al-Kateb, H., Bähring, S., Hoffmann, K., Strauch, K., Busjahn, A., Nürnberg, G., Jouma, M., Bautz, E.K., Dresesl, H.A., and Luft, F.C. (2002) Mutation in the ARH gene and a chromosome 13q locus influence cholesterol levels in a new form of digenic- recessive hypercholesterolemia. Circ Res 90, 951-8. The probes to BACs on chromosome 12p have been labeled with red, green, and yellow flourescence. The figure shows that in these examples the markers are not in the Knoblauch, H., Bauerfeind, A., Krähenbühl, C., Daury, A., same order on the two copies of chromosome 12p in the cells. This finding indicates that a rearrangement has taken place. Rohde, K., Bejanin, S., Essioux, L., Schuster, H., Luft, F.C., and Reich, J. (2002) Common haplotypes in six lipid genes explain forty percent of the genetic variance in the general population. Hum Mol Genet 11, 1477-1485. Preeclampsia Müller, D.N., Shagdarsuren, E., Dechend, R., Hampich, F., Ralf Dechend (Cardiology Section) is following a fascinating Park, J-K., Fiebeler, A., Schmidt, F., Theuer, J., Bieringer, M., line of evidence involving activating AT1 receptor antibodies Viedt, C., Kreuzer, J., Heidecke, H., Mervaala, E., Haller, H., that appear before clinical signs of preeclampsia and that and Luft, F.C. (2002) Immunosuppressive, anti-inflammatory disappear after delivery. The group recently studied the treatment ameliorates angiotensin II-induced renal damage. effects of these autoantibodies on trophoblasts and vascular Am J Pathol 161, 1679-1693. smooth muscle cells in terms of reactive oxygen species production. They convincingly showed that the antibodies activate the NADH oxidase in these cells and that the result- Structure of the Group ing reactive oxygen species stimulate the transcription factor NF-B. As a result, the cells produce various factors (e.g., Group Leader tissue factor) that could contribute to the development of pre- Prof. Dr. Friedrich C. Luft eclampsia. Their work also raises potentially important therapeutic possibilities. An animal model is being developed. Scientists Dr. Sylvia Bähring Dr. Andreas Busjahn (HealthTwiSt GmbH) Milestones Prof. Dr. Ralph Kettritz Dr. Volkmar Gross Arya M. Sharma has left the Nephrology/Hypertension Dr. Dominik N. Müller section to assume a new position as director of a department Dr. Anette Fiebeler for obesity studies at McMasters University, Ontario, Canada. Dr. Volker Homuth His duties will be assumed by Jens Jordan (Helmholtz Dr. Ralf Dechend (Cardiology Section) fellow), who has been appointed a Professor of Medicine (Clinical Pharmacology) at the Charité. Maik Gollasch Doctoral Students (Helmholtz fellow) will assume new duties as Associate Pro- Hussam Al-Kateb fessor of Physiology at the Louisiana State University, New Atakan Aydin Orleans, LA, USA. Both Arya Sharma and Maik Gollasch Erdenechimeg Shagdarsuren will maintain active associations with the Nephrology/Hyper- tension section in Berlin. Ralph Kettritz was recently appoint- Technical Assistants ed a Professor of Medicine (Nephrology) at the Charité. Sabine Grüger Ilona Kamer Christine Junghans Selected Publications Eireen Klein Astrid Mühl Obst, M., Gross, V., Janke, J., Schneider, W., and Luft, F.C. Yevette Neuhaus (2003) Pressure natriuresis in AT2-receptor deficient mice Regina Uhlmann with L-NAME hypertension. J Am Soc Nephrol 14, 303-10. Susanne Rolle

Dechend, R., Müller, D.N., Viedt, C., Wallukat, G., Park, Manager of Sponsored Programs J-K., Theuer, J., Barta, P., Homuth, V., Fiebeler, A., Kreuzer, Suzanne Wissler J., Dietz, R., Haller, H., and Luft, F.C. (2003) AT1 receptor antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation 107, 1632-9.

Choi, M., Rolle, S., Wellner, M., Scheidereit, C., Cardoso, M.C., Luft, F.C., and Kettritz, R. (2003) Inhibition of NF-B 26

Control of Smooth Muscle Cell Control of arterial tone by perivascular fat Function Virtually all blood vessels are surrounded by variable amounts of adipose tissue. Based on our results, we suggest Maik Gollasch (Helmholtz Fellow) that perivascular fat elaborates an adventitium-derived relaxing factor (ADRF). Release of ADRF is Ca2+-dependent and is regulated by intracellular signaling pathways involving tyrosine kinase and protein kinase A. In small mesenteric arteries, perivascular adipose tissue induces vasorelaxation by activating smooth muscle delayed-rectifier K+ channels. In collaboration with Dr. W.-H. Schunck’s group, we investigated the effects of P450-dependent epoxygenation of eicosa- pentaenoic acid on potassium channels. In collaboration with Drs. A. Otto and E.-C. Müller, we are currently examining the identity of “ADRF”. Perturbations of ADRF release and function could conceivably contribute to obesity-induced hypertension and to the development of arterial dysfunction in obesity and in other chronic vessel diseases.

Transport properties of INDY

The work in our lab focuses on the ionic mechanisms respon- Using two electrode voltage clamp and flux measurements in sible for the onset and maintenance of intrinsic (myogenic) Xenopus oocytes, we have been able to show that the life- vascular tone of small arteries. A second area of research is extending gene Indy encodes an exchanger for Krebs-cycle directed towards identifying the role of the perivascular fat as intermediates. We propose that the effect of decreasing Indy a modulator of arterial tone, with specific emphasis on the activity, as in long-lived Indy Drosophila mutants, may be to resistance vasculature. A third area of research focuses on the alter energy metabolism in a manner that favors life span dicarboxylate transporter encoded by the life-extending gene extension. Current work examines transport mechanisms of Indy (“I’m not dead yet”). Indy for tricarboxylates and Indy homologs in humans.

Calcium sparks and control of myogenic tone

Recent evidence indicates a role for subcellular calcium sparks as negative feedback regulators of arterial tone. Cal- Calcium spark and spontaneous transient outward current (STOC) activity in cium sparks result from the concerted opening of a few ryan- freshly-isolated wt and BKalpha-/- cerebral arterial cells. odine-sensitive calcium channels (RyR) in the sarcoplasmic (A) STOCs were recorded at increasing membrane potentials, and STOC frequency at –20mV from 5-7 wt and BKalpha-/- cells. (B) Confocal line-scans of fluo-3-loaded wt reticulum. We use a combined approach, utilizing single cell and BK-/- cerebral arterial cells, and time-course of corresponding Ca2+ sparks. Spark isolation, ion channel recording techniques, and intracellular amplitudes were measured as local fractional fluorescence increases (F/Fo; Fo is baseline). Spark duration was measured at half-maximal amplitude; n=5-12 randomly sel- calcium as well as calcium spark measurements using con- ected cells per genotype. All data are means ± SEM; *P<0.05; **P<0.01. ventional fluorescent imaging, and confocal laser scanning microscopy, diameter and membrane potential measurements in intact pressurized arteries, and expression of ion channels. Using gene knockout animals, we have been able to show that the ß1-subunit (BKß1) of the large-conductance calcium- activated potassium (BK) channel represents the molecular sensor of calcium sparks to reduce myogenic tone. Deletion of BKß1 disrupts coupling between calcium sparks and BK channels, leading to increased arterial tone and systemic blood pressure in mice. We have shown that variants in the gene (KCNMB1) coding for the BKß1 subunit are associated with baroreflex function in humans. Current work examines the role of canonical Transient Receptor Potential Channels (TRPC6, TPRC1) and L-type channels in release of calcium sparks and the role of the pore-forming BKalpha subunit in control of vascular tone. Malfunction of the calcium spark/ BK channel pathway may be an important mechanism for the development of human hypertension. 27

Selected Publications

Dubrovska, G., Verlohren, S., Luft, F.C., Gollasch, M.: Mechanisms of ADRF release from rat aortic adventitial adipose tissue. (2004). Am. J. Physiol. 286(3):H1107-13.

Lauterbach, B., Barbosa-Sicard, E., Wang, M.H., Honeck, H., Kärgel, E., Theuer, J., Haller, H., Luft, F.C., Gollasch, M., Schunck, W.H. (2002). Cytochrome P450-dependent eicosa- pentaenoic acid metabolites are novel BK channel activators. Hypertension. 39 (2):609-613.

Löhn, M., Muzzulini, U., Conrad, H., Kirsch, T., Litteral, J., Waldron, P., Klugbauer, N., Hofmann, F., Haller, H., Luft, F.C., Huang Yu, Gollasch, M. (2002). Cilnidipine is a novel slow-acting blocker of vascular L-type calcium channels that does not target protein kinase C. J. Hypertens. 20:885–893.

Gollasch, M., Tank, J., Luft, F.C., Jordan, J., Maass, P., Krasko, C., Sharma, A.M., Busjahn, J., Bähring, S. (2002). The BK channel ß1 subunit gene is associated with human baroreflex and blood pressure regulation. J. Hypertension 20:927–933.

Löhn, M., Dombrovska, G., Lauterbach, B., Luft, F.C., Gollasch, M., Sharma, A.M. (2002). Periadvential fat releases a vascular relaxing factor. FASEB J. 16: 1057-1063.

Structure of the Group

Group Leader PD Dr. Maik Gollasch

Postdoctoral Fellows Dr. Galyna Dubrovska Dr. Nilufar Mohebbi

Graduate Students Kirill Essin Stefan Verlohren Felix Knauf Carsten Teichert Gabor Fesüs

Technical Assistant Diana Herold 28

Molecular Mechanisms of the clinical use of norepinephrine transport inhibitors. We partici- Metabolic Syndrome pated in studies on the pathophysiology of monogenic hypertension and brachydactyly. Sylvia Bähring’s group showed that the syndrome is caused by a complex genetic Jens Jordan (Helmholtz Fellow) rearrangement. We used systemic and intra-arterial infusions of various pharmacological agents to confirm or exclude candidate genes, such as PDE3A. Venous dysfunction contributes to many common cardiovascular diseases, such as vari- cosis, thrombosis, orthostatic intolerance, and, perhaps, arterial hypertension. In our twin studies, we found evidence for a strong genetic influence on venous function. Currently, we are analyzing the heritability of metabolic parameters, such as free and bound leptin and adiponectin concentrations.

Adipose tissue and the pathogenesis of obesity- associated cardiovascular disease

Adipose tissue secretes a large number of products that have been implicated in the pathogenesis of cardiovascular disease. We are particularly interested in adipose tissue derived angiotensin II and leptin. Leptin circulates in both a receptor-bound The main interest of the group is both mechanism- and and in a free form. Bound and free leptin appear to have patient–oriented research in the field of clinical autonomic different biological functions. We found a strong correlation disorders, arterial hypertension, obesity, and the metabolic between sympathetic nerve traffic and bound leptin concen- syndrome. In the last few years, we established a clinical trations in normal weight men. In contrast, sympathetic research center (CRC) to provide the infrastructure for these activity was not related to free leptin concentrations. The studies. One intention of our group is to combine patient physiological role of angiotensin II in adipose tissue is poorly oriented research with basic science and genetics in the field understood. We applied angiotensin II to the interstitial space of cardiovascular and metabolic diseases. The purpose of our in both adipose tissue and skeletal muscle using the micro- research is to develop new treatment strategies for patients dialysis technique. Remarkably, we did not see a major change with obesity (also called “metabolic syndrome”), orthostatic in tissue blood flow. Yet, angiotensin II changed lipid and intolerance, autonomic failure, and neurogenic hypertension carbohydrate metabolism in a tissue-specific fashion. The based on a better understanding of the pathophysiology of metabolic effect of angiotensin II might contribute to insulin these clinical syndromes. We believe that studies on rare resistance and explain, in part, the beneficial effect of angio- human diseases that are associated with low blood pressure tensin II inhibition on carbohydrate metabolism. We are may also give important insight into the mechanisms of continuing these studies in obese patients with and without essential and obesity associated hypertension. To elucidate arterial hypertension. We combine pharmacological methods, the potential influence of candidate genes we conducted twin functional metabolic studies, and adipose tissue gene expres- studies. We have a close collaboration with other groups at sion analysis. These studies are conducted in close collabo- the MDC to confirm hypotheses that are generated in humans ration with the adipocytes biology laboratory (Dr Engeli and in already available or in newly created animal models. colleagues).

Genetic influences on cardiovascular and Selected Publications metabolic regulation in health and disease Tank J., Schroeder C., Diedrich A., Szczech E., Haertter S., In earlier studies, we found a functional mutation in the nore- Sharma A.M., Luft F.C., and Jordan J. (2003), Selective im- pinephrine transporter gene in patients with familial ortho- pairment in sympathetic vasomotor control with norepi- static intolerance. We further elucidated the role of the nore- nephrine transporter inhibition Circulation 107, 2956-61. pinephrine transporter in cardiovascular and metabolic regulation in a series of human pharmacological experiments. Tank J., Jordan J., Diedrich A., Schroeder C., Stoffels M., We found that norepinephrine transporter inhibition causes a Franke G., Sharma A.M., Luft F.C., and Brabant G. (2003) selective impairment in sympathetic vasomotor regulation, Bound leptin and sympathetic outflow in nonobese men. which is suggestive of a central nervous sympatholytic effect. Journal of Clinical Endocrinology and Metabolism 88, The baroreflex impairment resulted in hypersensitivity to 4955-9. vasoactive drugs. Furthermore, norepinephrine transporter inhibition elicited metabolic changes, both systemically and Schröder C., Tank J., Boschmann M., Diedrich A., Sharma at the adipose tissue level. In particular, we found impaired A.M., Biaggioni I., Luft F.C., Jordan J. (2002) Selective nore- lipid utilization. Our observations attest to the importance of pinephrine reuptake inhibition as a human model of ortho- the norepinephrine transporter in metabolic and cardiovascu- static intolerance. Circulation 105, 347-353. lar regulation. They may have important implications for the 29

Jordan J., Tank J., Shannon J.R., Diedrich A., Lipp, Schröder C., Arnold G., Sharma A.M., Biaggioni I., Robertson D., and Luft F.C. (2002) Baroreflex buffering and susceptibility to vasoactive drugs. Circulation 105, 1459-1464.

Boschmann M., Schroeder C., Christensen N.J., Tank J., Krupp G., Biaggioni I., Klaus S., Sharma A., Luft F.C., and Jordan J. (2002) Norepinephrine transporter function and autonomic control of metabolism. Journal of Clinical Endo- crinology and Metabolism 86, 2803-2810.

Structure of the Group

Group Leader Prof. Dr. Jens Jordan

Scientists Dr. Michael Boschmann PD Dr. Karsten Heusser Dr. Heidrun Mehling Dr. Christoph Schröder Dr. Jens Tank

Doctoral Students Frauke Adams Andreas Birkenfeld Petra Budziarek

Study Nurses/Technical Assistants Gabriele Franke Iris Gottschalk Nadine Krüger Grit Stoffels Anke Strauss Elke Szczech

Manager of Sponsored Programs Susanne Wissler 30

Functional Characterization leads to a strong decrease in regenerative sprouting of neuri- of Newly Identified Human Importin tes from lesioned neurons. The data obtained support a model Proteins whereby importin , which is newly synthesized in injured neurons, mediates in complex with importin and the motor protein dynein retrograde transport of signaling proteins from Matthias Köhler (Helmholtz Fellow) the injury site to the axoplasm, regulating repair mechanisms of lesioned nerves. Beate Friedrich, Christina Quensel, and Tanja Schmidt, doctoral and postdoctoral fellows in our group, are working on the identification of specific functions of the importins in living cells and organisms.

Selected Publications

Melén, K., Fagerlund, R., Franke, J., Köhler, M., Kinnunen, L., and Julkunen, I (2003). Importin nuclear localization signal binding sites for STAT1, STAT2, and Influenza A virus nucleoprotein. J. Biol. Chem. 278, 28193-28200

Hanz, S., Perlson, E., Willis, D., Zheng, J., Massarwa, R., Huerta, J.J., Koltzenburg, M., Köhler, M., Minnen, J., Twiss, J.F., and Fainzilber, M. (2003). Axoplasmic importins enable Our group is working on nucleocytoplasmic protein transport. retrograde injury signaling in lesioned nerve. Neuron 40 (6): We have identified, in collaboration with Enno Hartmann and 1095-1104 Dirk Görlich, several novel human isoforms of the importin protein family, which mediate nucleocytoplasmic protein Köhler, M., Fiebeler, A., Hartwig, M., Thiel, S., Prehn, S., import in complex with importin . Using in-vitro import Kettritz, R., Luft, F.C., and Hartmann, E. (2002). Differential assays, we have elucidated the import pathways of different expression of classical nuclear transport factors during cellu- import substrates and could demonstrate that the importins lar proliferation and differentiation. Cell. Physiol. Biochem. differ in their substrate specificity in-vitro. Our group is 12, 335-344. further working on the identification of the in-vivo relevance of the different importins. Our doctoral fellows, Maite Hartwig and Sebastian Thiel, showed that the importins are Structure of the Group differentially expressed in various models of cellular proliferation and differentiation. In collaboration with the group of Group Leader Ilkka Julkunen from Helsinki, Finland, our doctoral fellow, Dr. Matthias Köhler Jacqueline Franke, investigated the import mechanisms of STAT proteins. Together, we demonstrated that activated Scientists STATs’1 and 2 strongly bind to importin 5 but not to other Dr. Christina Quensel importins. Furthermore, we identified the nuclear localization Dr. Tanja Schmidt binding sites for STAT1, STAT2 and influenza Avirus nucleoprotein on importin 5. A recent collaboration with the group Graduate Students of Mike Fainzilber, Weizmann Institute of Science, Rehovot, Beate Friedrich Israel, investigated the role of the importins in retrograde transport of signaling molecules in injured neurons. We Technical Assistants showed that inhibition of the importin / transport complex Brigitte Nentwig

Impairment of in-vitro regenerative neuronal outgrowth by inhibition of importin / meditated retrograde transport of signaling proteins via excessive addition of NLS- peptides. 31

Obesity and Hypertension derived relaxing factor that acts by tyrosine kinase-dependent activation of K+ channels in vascular smooth muscle cells. Maik Gollasch and his group are following up on these Arya M. Sharma studies (see report by M. Gollasch).

Adipocytes, preadipocyte differenitation and angiotensin (Ang) II

Recent studies suggest that Ang II plays a role in the adipogenesis of murine preadipocytes. We isolated preadipocytes from human adipose tissue and stimulated them to differentiate. Quantitation of gene expression during adipogenesis was performed for renin-angiotensin system genes. We added angiotensinogen, Ang II, or angiotensin receptor antagonists to the differentiation medium. We also examined the influence of adipocytes on adipogenesis by co-culture experiments. Stimulation of the Ang II type 1 receptor by Ang II reduced adipose conversion, whereas blockade of this receptor markedly enhanced adipogenesis. Adipocytes were able to inhibit preadipocyte differentiation in the co-culture. The effect was abolished by blockade of the Ang II type 1 recep- Summary tor. This finding suggested a functional role of the renin- angiotensin system in the differentiation of human adipose Obesity is the most rapidly growing public health problem tissue. Because angiotensinogen secretion and Ang II genera- worldwide. The condition commonly leads to cardiovascular tion are characteristic features of adipogenesis, we postulated disease and features the well-known risk factors of hyper- a paracrine negative-feedback loop that inhibits further tension, diabetes mellitus (metabolic syndrome), and lipid recruitment of preadipocytes by maturing adipocytes. Our disturbances. In many developed countries, thirty to forty hypothesis is supported by in vivo human studies. percent of the population is overweight or obese. In the Uni- ted States, this number is currently about 50% and is growing yearly. The condition is strongly influenced by genetic Adiponectin and inflammation variance. Adipose tissue has revealed itself as amazingly diversified in terms of producing cytokines, chemokines, and Low plasma levels of the adipocyte-produced anti-inflamma- hormones, as well as being a target for these molecules. Our tory factor adiponectin characterize obesity and insulin resi- group has directed its research efforts at elucidating the stance. To elucidate the relationship among plasma levels of relationship between obesity and cardiovascular disease. adiponectin, adiponectin gene expression in adipose tissue, and markers of inflammation, we obtained blood samples, anthropometric measures, and subcutaneous adipose tissue Adventitia-derived relaxing factor samples from 65 postmenopausal healthy women. Adiponec- tin plasma levels and adipose-tissue gene expression were Virtually all blood vessels are surrounded by adventitial fat. significantly lower in obese subjects and inversely correlated Adipocytes produce a host of vasoactive substances that may with obesity-associated variables, including high-sensitive influence vascular contraction. We tested whether perivascu- C-reactive protein (hs-CRP) and interleukin-6 (IL-6). Despite lar adipose tissue modulates contraction of aortic ring prepa- adjustment for obesity-associated variables, plasma levels of rations. We studied aortic rings surrounded by periadventitial adiponectin were significantly correlated with adiponectin adipose tissue from adult Sprague-Dawley rats. At maximum gene expression. Furthermore, the inverse correlation concentrations of angiotensin II, serotonin, and phenyl- between plasma levels of hs-CRP and plasma adiponectin ephrine, the contractile response of intact rings was, respect- remained significant despite correction for obesity-associated ively, 95%, 80%, and 30% lower than that of vessels without variables, whereas the inverse correlation between adiponec- periadventitial fat. The anticontractile effect of periadventitial tin plasma levels or adiponectin gene expression in adipose fat was reduced by inhibition of ATP-dependent K+ channels tissue with plasma IL-6 were largely dependent on the with glibenclamide and by the tyrosine kinase inhibitor geni- clustering of obesity-associated variables. In conclusion, our stein. Blocking NOS, cyclo-oxygenase, cytochrome P450, or data suggest a transcriptional mechanism leading to decreas- adenosine receptors did not restore the vascular response in ed adiponectin plasma levels in obese women and demon- intact vessels. The anticontractile effect of perivascular fat strate that low levels of adiponectin are associated with was present in Zucker fa/fa rats, suggesting that leptin recep- higher levels of hs-CRP and IL-6, two inflammatory mediators were not responsible. Transferring the bath solution from tors and markers of increased cardiovascular risk. Stefan intact vessels, isolated periadventitial tissue, and cultured rat Engeli is currently responsible for the adipocyte group. They adipocytes to precontracted vessels lacking periadventitial fat are investigating the regulation of adiponectin and other resulted in a rapid relaxation. We suggest that perivascular adipocyte products in obese persons before and after weight adventitial adipose tissue releases a transferable adventitium- loss. 32

ABC D

unstimulated stimulated stimulated stimulated - - adipocytes adipocytes - - - blockade Undifferentiated preadipocytes (A) do not store lipids, but when stimulated (B) they differentiate and synthesize triglycerides. When exposed to Ang II (C), preadipocytes do not differentiate. Blocking the actions of Ang II by an Ang II receptor blocker (D) allows adipocyte differentiation to occur.

Pharmacological treatment of obesity Janke, J., Engeli, S., Gorzelniak, K., Luft, F.C., and Sharma, A.M. (2002) Mature adipocytes inhibit in vitro differentiation Sibutramine, a serotonin and norepinephrine transporter of human preadipocytes via angiotensin type 1 receptors. blocker, is widely used as an adjunctive obesity treatment. Diabetes. 51, 1699-707 However, its effect on cardiovascular regulation is imperfect- ly defined. We collaborated with Jens Jordan’s group to eluci- Sharma, A.M., Janke, J., Gorzelniak, K., Engeli, S., and Luft, date sibutramine’s actions. In 11 healthy subjects, we compa- F.C. (2002) Blockade of the renin-angiotensin system red the effect of sibutramine or matching placebo on prevents type 2 diabetes by promoting the formation of new cardiovascular responses to autonomic reflex tests and to a fat cells. Hypertension 40, 609-611. graded head-up tilt test. In addition, we tested sibutramine in combination with metoprolol. Testing was conducted in a Birkenfeld, A., Schroeder, C., Boschmann, M., Tank, J., double blind and crossover fashion. Sibutramine increased Franke, G., Luft, F.C., Sharma, A.M., and Jordan, J. (2002) upright blood pressure and upright heart rate. This effect was Effect of sibutramine on autonomic cardiovascular regula- abolished with metoprolol. The blood pressure response to tion. Circulation 106, 2459-2465. cold pressor and handgrip testing was attenuated with sibutramine compared with placebo. Sibutramine also decreased low-frequency oscillations of blood pressure and plasma Structure of the group norepinephrine concentrations in the supine position. Our study showed that the cardiovascular effect of the antiobesity Group Leader drug sibutramine results from a complex interaction of Prof. Dr. Arya M. Sharma peripheral and central nervous system effects. The inhibitory clonidine-like action of sibutramine on the central nervous Scientists system attenuates the peripheral stimulatory effect. Dr. Stefan Engeli Dr. Jürgen Jahnke Dr. Maren Wellner Milestones Dr. Kerstin Gorzelniak

Arya Sharma has assumed new duties as chairman of a Doctoral Students department for Obesity Research at McMasters University, Mareike Feldpausch Hamilton, Ontario, Canada. However, obesity related hyper- Frauke Hartwig tension continues to be a research focus of the Nephrology/ Hypertension section of the Franz Volhard Clinic. Jens Jordan Study Nurses will assume responsibility for the group. A close relationship Iris Gottschalk between Arya Sharma and the Nephrology/Hypertension Anke Strauss section is being maintained. Dietitians Jana Böhnke Selected publications Technician Engeli, S., Feldpausch, M., Gorzelniak, K., Hartwig, F., Henning Damm Heintze, U., Janke, J., Luft, F.C., and Sharma, A.M. (2003) Adiponectin gene expression, insulin sensitivity, and inflam- Manager of Sponsored Programs matory markers in obese women. Diabetes 52, 942-947. Suzanne Wissler

Löhn, M., Dubrowska, G., Lauterbach, B., Luft, F.C., Gollasch, M., and Sharma, A.M. (2002) Periadvential adipose tissue releases a vascular relaxing factor. FASEB J 16, 1057-63. Heart Disease

Cardiovascular Molecular Genetics large family with DCM also linked to CMD1G at chromosome 2q31, a titin missense mutation, W930R, is predicted to disrupt a highly conserved hydrophobic core sequence of an Ludwig Thierfelder immunoglobulin fold located in the Z-disc/I-band transition zone. The identification of mechanisms of titin mutations should provide further insights into the pathogenesis of familial forms of congestive heart failure and myofibrillar titin turnover.

Isolated non-compaction of the left ventricle in the Familial forms of heart failure adult

Congestive heart failure is a complex syndrome resulting Isolated non-compaction of the left ventricle (INVC) is a rare from various disease states with inadequate cardiac output. disorder characterized by wide intertrabecular spaces due to Familial forms of congestive heart failure can be studied by an arrest of endomyocardial morphogenesis. It is well-known genetic analyses. Cardiomyopathies (CMPs) are heart muscle that infantile INVC is an X-chromosomal disease and caused disorders with a strong genetic component. The most by mutations in G4.5, a gene with a yet unknown function. common CMP, dilated cardiomyopathy (DCM), is caused by We study a large population of adult INVC patients to assess autosomal dominant mutations in 20-30% of cases. A number whether genetic defects can also be be accounted for in this of DCM disease genes have been identified that fall into population. In one large pedigree, INVC segregated as an different functional classes. This suggests that different autosomal dominant trait and an autosomal locus was identi- disease pathways can culminate in the development of conge- fied in a genomewide linkage analysis. Analyses of the stive heart failure due to progressive myocardial failure. putative disease gene are underway.

Little is known about the prevalence of mutations in individual DCM disease genes. In two DCM families, positional Malignant ventricular arrhythmias in a large cloning efforts in our laboratory have recently led to the Canadian founder population identification of mutations in titin (TTN) causing one form of non-syndromic DCM. Titin is the largest known molecule in Arrhythmogenic right ventricular cardiomyopathy (ARVC) is mammals (3-3.7MDa) and encoded by a cDNA of up to a difficult-to-diagnose cardiac condition associated with 100kb. A truncation mutation of A-band titin and a missense sudden death and heart failure. We have identified an auto- mutation in I-band titin cause a similar phenotype in two un- somal dominant founder mutation on chromosome 3p25 in a related families. Interestingly, although both mutations are large Canadian cardiomyopathy population of circa 400 indi- expressed in cardiac and skeletal muscle, only cardiac muscle viduals at a 50% risk of inheriting the condition. ARVC in is clinically affected. Titin molecules extend from sarcomeric these individuals is associated with a distinct electrocardio- Z-discs to M-lines, provide an extensible scaffold for the graphic pattern and a life expectancy of males of less than 40 contractile machinery and are critical for myofibrillar elasti- years. Mutational analyses of approximately 15 genes located city and integrity. in a 3Mbp region on chromosome 3p25 are in progress.

In a large DCM kindred, a segregating 2bp insertion mutation in titin exon 326 causes a frame shift, thereby truncating Molecular genetics of pseudoxanthoma elasticum A-band titin. The truncated ≈2MDa protein is expressed in (PXE) skeletal muscle but Western blot studies with epitope-specific anti-titin antibodies suggest it undergoes proteolytic process- Pseudoxanthoma elasticum (PXE) is a heritable systemic ing into a 1.14MDa subfragment by site-specific cleavage disorder of the elastic tissue characterized by degenerative within the PEVK region. Interestingly, in a cardiac biopsy calcification with subsequent disintegration and destruction sample taken from an affected patient, the truncation of the elastic tissue of several organs. Cardiovascular disease mutation appears not to be expressed (or actively degraded) at encompasses a wide clinical spectrum from mental fatigue the cDNA or protein level. A mouse model expressing the syndrome to early cardiovascular death due to myocardial truncation mutation should provide further insight. In another infarction or, very rarely, gastrointestinal hemorrhage. We 34

Seven pedigrees from a large Newfoundland population suffering from an autosomal dominant cardiomyopathy causing early sudden cardiac death, especially in men. All affected individuals share a founder mutation on chromosome 3p25.

have mapped the PXE locus to a 500 kb interval on chromo- Structure of the Group some 16p13.1 and have shown that mutations in a transmembrane transporter protein, ABC-C6 (also known as MRP-6), Group Leader cause PXE. Prof. Dr. Ludwig Thierfelder

Scientists Selected Publications Dr. Sabine Sasse-Klaassen Dr. Bertold Struk Gerull B, Gramlich M, Atherton J, McNabb M, Trombitás K, Dr. Jörg Drenckhahn Sasse-Klaassen S, Seidman JG, Seidman CE, Granzier H, Labeit S, Frenneaux M, Thierfelder L.(2003). Mutations of Graduate Students TTN, encoding the giant muscle filament titin, cause familial Kalina Ramadanova dilated cardiomyopathy. Nat Genet 30(2):201-4. Technical Assistants Sasse-Klaassen S, Gerull B, Oechslin E, Jenni R, Thierfelder Sigrid Milan L. (2003). Isolated noncompaction of the left ventricular Tina Hagena* myocardium in the adult is an autosomal dominant disorder in the majority of patients. Am J Med Genet. Jun 1;119A * part of the time reported (2):162-7.

Cai L, Struk B, Adams MD, et al. (2000). A 500-kb region on chromosome 16p13.1 contains the pseudoxanthoma elasticum locus: high-resolution mapping and genomic structure. J Mol Med. 78(1):36-46.

Struk B, Cai L, Zach S, Ji W, et al. (2000). Mutations of the gene encoding the transmembrane transporter protein ABC-C6 cause pseudoxanthoma elasticum. J Mol Med. 78(5):282-6. 35

Genetic Disorders of largely unknown whether genetic factors play a role in this the Cardiovascular System complex syndrome. We have identified a large kindred (referred to as ‘Kindred A’) with multiple members clinically affected by one or several features of APS. Segregation, Brenda Gerull (Helmholtz Fellow) linkage, and molecular analyses have identified at least two genetic defects in Kindred A, one being a factor V Leiden mutation in a nuclear pedigree of Kindred A and a yet unknown defect on chromosome 10p12 causing thrombozytopenia. Other APS features in Kindred A (presence of antipho- pholipd antibodies; stillbirth, etc.) have not been mapped yet. We are currently screening for the chromosome 10p12 gene carrying the mutation responsible for thrombozytopenia in Kindred A.

Selected Publications

Sasse-Klaassen S, Gerull B, Oechslin E, Jenni R, Thierfelder L. (2003) Isolated noncompaction of the left ventricular myocardium in the adult is an autosomal dominant disorder in the majority of patients. Am J Med Genet. 1;119A(2):162-7.

Titin’s role in heritable cardiac and skeletal muscle Gerull B, Gramlich M, Atherton J, McNabb M, Trombitás K, disorders Sasse-Klaassen S, Seidman JG, Seidman CE, Granzier H, Labeit S, Frenneaux M, Thierfelder L. (2002) Mutations of TTN encodes for the largest known protein, titin. Titin serves TTN, encoding the giant muscle filament titin, cause familial as a scaffold in the sarcomere, plays a role in myofilament dilated cardiomyopathy. Nat Genet 30(2):201-4. turnover, and probably, in myocyte signal transduction. More than 360 exons code for multiple, alternatively spliced isoforms of approximately 1-3MDa in size which are expressed Structure of the Group in cardiac and skeletal muscle. A role for titin in non-muscle tissue (where it may be expressed at low levels) is less clear. Group Leader Dr. Brenda Gerull Several inherited human muscle disorders have been mapped to the TTN locus on chromosome 2q31. One form of dilated Scientists cardiomyopathy (CMD1G), tibial muscular dystrophy Dr. Arnd Heuser (TMD), and proximal myopathy with respiratory failure (all Dr. Beate Michely autosomal dominant disorders) have been mapped to the TTN locus on chromosome 2q31. For CMD1G and TMD, TTN mu- Technical Assistant tations have been identified by us and others. One of the Ilona Trippmacher CMD1G mutations is a complex mutation causing a frame shift in A-band titin (thereby introducing a premature stop codon) and, depending on muscle tissue, different posttranscriptional modifications of the truncated protein. In skeletal muscle, the truncated titin (expected size approximately 2MDa) is proteolytically digested to a 1,14MDa protein containing Z-disc and (partial) I-band titin. The truncated titin, however, is not present in a cardiac biopsy sample from an affected family member suggesting differences in posttranscriptional modifications. The question of whether the human cardiac phenotype in CMD1G is due to haploinsufficiency (as suggested by the Western blot results) and why no clinical phenotype of skeletal muscle is observed remain uncertain.

Genetics of antiphospholipid antibody syndrome and thrombozytopenia linked to chromosome 10p12

The antiphospholipid antibody syndrome (APS) is a complex, usually aquired hypercoagulation disorder, clinically characterized by thromboembolism, stillbirth, and thrombozytopenia in the presence of antiphospholipid antibodies. It is 36

Myocardial Regeneration

Rainer Dietz

When fully differentiated, the mammalian heart is composed of cardiomyocytes which have withdrawn from the cell cycle. Thus, the heart is not able to compensate for cell loss render- ing it biologically inert in regard to regeneration. So far, conventional therapy given to patients with heart failure aims at the reduction of the hemodynamic load in order to alleviate cardiac work. The establishment of a molecular approach to reinstall cardiomyocyte cell division would revolutionize standard treatment regimens for heart failure patients.

In general, there are two possibilities to prevent loss of car- Ectopic E2F2 induces cell cycle reentry and mitosis in cardiomyocytes. Cardiomyocytes were infected with adenovirus wild-type E2F2 (100 pfu/cell) and continued to be culti- diac contractile tissue after myocardial damage: 1) prevention vated for 48 hours. For the detection of M-phase specific phosphorylation of Histone of cell death, and 2) reinduction of cell cycle activity in sur- H3, fixed cells were stained with rabbit polyclonal antibody to phospho-Histone H3, which recognizes serine 10 phosphorylation of Histone H3 (green). Cardiomyocytes rounding healthy cardiomyocytes. Using mainly cell culture were identified by costaining for the expression of MHC (MF20, red), and genomic DNA modells of primary cardiomyocytes our group tries to identify (Hoechst 33258, blue). Description of mitotic figures identified in cardiomyocytes: A, pro- as well as anti-apoptotic signalling pathways in different prophase; B, metaphase; C, early telophase; D, late telophase. forms of cardiomyocyte apoptosis. As a consequence of both acute and chronic myocardial damage, in most instances, detrimental cardiomyocyte hypertrophy develops. However, the intracellular pathways responsible for this myocardial pathways in cardiomyocytes. More importantly, this is the maladaptive growth still remain enigmatic. Therefore, the first observation of a pRb-independent mechanism regulating intercalation between pro- and anti-apoptotic pathways on E2F1-dependent transcription and apoptosis. one side and classical growth cascades including cell cycle pathways on the other is also in the focus of our research interest. Phosphorylation by protein kinase CK2: A signalling switch for the caspase-inhibiting protein ARC

Cardiomyocyte apoptosis requires cell cycle acti- ARC, a recently discovered anti-apoptotic factor, the express- vation and downregulation of cell cycle inhibitors ion of which appears to be restricted to cardiac and skeletal muscle tissue, was found by our group to be a substrate of the We have found that both cyclin-dependent kinase inhibitors casein kinase II (CK2). Constitutive phosphorylation of ARC p21CIP1 and p27KIP1 need to be downregulated in order to by CK2 is required for ARC to act in an anti-apoptotic trigger apoptosis in cardiomyocytes. Also cardiomyocyte fashion. apoptosis is characterized by activation of the pRb/E2F-dependent pathway. Moreover, yeast two hybrid screening of a human heart library revealed that the transcription factor p21CIP1 controls proliferating cell nuclear antigen E2F1, which previously has been shown by us and other protein level in adult cardiomyocytes groups to act in a pro-apoptotic fashion in primary cardiomyocytes, interacts with the ETS-related transcription factor While trying to understand how cell death of cardiomyocytes GABP1. This interaction links growth and cell death related is triggered, much effort of our group is devoted to decipher- 37

ing the regulation of cardiomyocyte cell cycle withdrawal. Employing different models, we have found that p21CIP1 plays a critical role in the prevention of cardiomyocyte cell cycle activation. Importantly, p21CIP appears to act as a suppressor on cardiomyocyte cell cycle by regulating the degradation of proliferating cell nuclear antigen (PCNA) rather than by its inhibitory effect on cyclin-dependent kinases.

Selected Publications

Engel FB, Hauck L, Boehm M, Nabel EG, Dietz R, von Harsdorf R (2003): p21CIP1 controls proliferating cell nuclear antigen protein level in adult cardiomyocytes. Mol Cell Biol, 23: 555-565

Hauck L, Kaba RG, Lipp M, Dietz R, von Harsdorf R (2002): Regulation of E2F1-dependent gene transcription and apoptosis by the ETS-related transcription factor GABPg1. Mol Cell Biol, 22: 2147-2158.

Li P, Li J, Müller EC, Otto A, Dietz R, von Harsdorf R (2002): Phosphorylation by protein kinase CK2: A signaling switch for the caspase inhibiting protein ARC. Mol Cell, 10: 247- 258.

Li J, Li P, Dietz R, von Harsdorf R (2002): Intracellular su- peroxide induces apoptosis in VSMCs: role of mitochondrial membrane potential, cytochrome C and caspases. Apoptosis, 7: 511-517.

Hauck L, Hansmann G, Dietz R, von Harsdorf R (2002): Inhibition of hypoxia-induced apoptosis by modulation of retinoblastoma protein-dependent signaling in cardiomyocytes. Circ Res, 91: 782-789

Structure of the Group

Group Leader Prof. Dr. Rainer Dietz

Scientists Prof. Dr. Rüdiger v. Harsdorf Dr. Ludger Hauck Dr. Peifeng Li Dr. Felix Mehrhof Dr. Stefan Donath Dr. Thomas Knaus Dr. Georg Hansmann* Dr. Junfeng An Dr. Jincheng Li*

Graduate and Undergraduate Students Technical Assistants Jana Bröcker Marlies Grieben Alan Punnoose Daniela Grothe Felix Engel* Janet Lips

* part of the time reported 38

Myocardial Nuclear Receptors in order to understand the mechanism of NF-B’s influence on Heart Failure heart remodeling.

Martin W. Bergmann (Helmholtz Fellow) CREB is essential for hypoxia/reoxygenation induced cardiomyocyte hypertrophy

Another set of experiments has focused on cardiomyocyte hypertrophy induced by hypoxia followed by reoxygenation similar to the ventricular remodeling observed in vivo after myocardial infarct. While hypertrophy was not altered by inhibiting NF-B activation, a role for CREB downstream of the PI3-kinase/AKT/GSK3 signaling pathway was identified in these studies. Interestingly, GSK3 did not alter CREB serine133 phosphorylation, the common endpoint of CREB stimulation regulating transactivation of CREB- responsive genes. Instead, GSK3 regulated CREB DNA binding, possibly by a second phosphorylation at CREB serine 129.

Statins protect cardiomyocytes from apoptosis by The group started in January 2001 focusing on the identifica- inactivating GSK3b tion of new targets in heart failure treatment by characterizing the signal transduction pathways leading to cardiomyocyte Our data imply differential sets of transcription factors invol- hypertrophy and apoptosis. We study the role of specific tran- ved in cardiac remodeling preceding heart failure. These scription factors employing both isolated neonatal and adult studies prompted us to investigate the effect of currently used cardiomyocytes as well as transgenic mice generated by drugs on cardiomyocyte nuclear signaling. Statins are used Cre/lox techniques. Previous studies by other groups had for their effect on cholesterol levels in blood. However, recent implicated transcription factor NF-AT as an important down- evidence suggests a direct effect on cardiac remodeling inde- stream molecule integrating Ca2+ – induced signaling towards pendent of vascular protection. Experiments with isolated rat cardiomyocyte hypertrophy. Similar to NF-AT, the transcrip- cardiomyocytes revealed activation of the well-known PI3- tion factor NF-B was first identified for its prominent role in kinase/AKT/GSK3 pathway resulting in reduced apoptosis. regulating inflammatory cytokines. However, NF-B was Downstream of GSK3, the transcription factor -catenin recently found to be important for B-cell proliferation in was stabilized as an effect of statin treatment. Hodgkins’s disease and shown to be necessary for G-protein coupled receptor – induced cardiomyocyte hypertrophy in vitro as well as survival of cardiomyocytes. Similarly, the Selected Publications transcription factor cAMP response element binding protein (CREB) is essential for cAMP-induced cytokine release as Bergmann, M. W., Staples, K. J., Smith, S. J., Barnes, P. J., well as cell proliferation and cardiomyocyte survival. and Newton, R. (2004) Glucocorticoid inhibition of GM-CSF from T cells is independent of control by NF-{kappa}B and CLE0. Am J Respir Cell Mol Biol, in press. NF-B is a suitable target to improve left ventricular remodeling in vivo Staples, K. J., Bergmann, M. W., Barnes, P. J., and Newton, R. (2003) Evidence for post-transcriptional regulation of inter- We have characterized the signaling pathways induced by leukin-5 by dexamethasone. Immunology 109, 527-535. Angiotensin II (a well known hypertrophy stimulus) in adult cardiomyocytes. In addition, mice with heart-specific NF-B Mehrhof, F. B., Muller, F. U., Bergmann, M. W., Li, P., Wang, inhibition were generated by mating -myosin heavy chain Y., Schmitz, W., Dietz, R., and von Harsdorf, R. (2001) In Cre-recombinase mice to loxP-IBN mice in collaboration cardiomyocyte hypoxia, insulin-like growth factor-I-induced with R. Schmidt-Ullrich, group Scheidereit. The mice have antiapoptotic signaling requires phosphatidylinositol-3-OH- been characterized at baseline as well as after 14 days of kinase-dependent and mitogen-activated protein kinase- AngII infusion by osmotic minipumps. Histologic, echocar- dependent activation of the transcription factor cAMP res- diography, and gene expression analysis revealed diminished ponse element-binding protein. Circulation 104, 2088-2094. hypertrophy in mice with heart-specific NF-B inhibition. Gene chip analysis comparing adult cardiomyocytes with Bergmann, M. W., Loser, P., Dietz, R., and von Harsdorf, R. adenoviral overexpression of NF-B inhibitor IBN to (2001) Effect of NF-kappa B Inhibition on TNF-alpha-indu- control virus transfected cells revealed a set of potential ced apoptosis and downstream pathways in cardiomyocytes. J NF-B targets, which seem to be heart specific as a control of Mol Cell Cardiol 33, 1223-1232. these genes by NF-B has not been described before. These target genes are currently validated by further experiments in 39

Newton, R., Staples, K. J., Hart, L., Barnes, P. J., and Berg- mann, M. W. (2001) GM-CSF expression in pulmonary epithelial cells is regulated negatively by posttranscriptional mechanisms. Biochem Biophys Res Commun 287, 249-253.

Staples, K. J., Bergmann, M., Tomita, K., Houslay, M. D., McPhee, I., Barnes, P. J., Giembycz, M. A., and Newton, R. (2001) Adenosine 3’,5’-cyclic monophosphate (cAMP)- dependent inhibition of IL-5 from human T lymphocytes is not mediated by the cAMP-dependent protein kinase A. J Immunol 167, 2074-2080.

Structure of the Group

Group Leader Dr. Martin W. Bergmann

Scientists Amina El Jamali* Anthony Baurand*

Graduate and Undergraduate Students Christian Freund Cindy Rechner* Josefine Bechstein* Hagen Kempf*

Technical Assistants Bärbel Pohl* Gabi Welsch* Silke Feineis* Anke Stollenwerk*

* part of the time reported 40

Cardiovascular Magnetic Perfusion of the peri-infarct myocardial tissue Resonance Andrew Taylor from the Baker Heart Research Institute, Melbourne, Australia spent one year in our group. He disco- Matthias G. Friedrich vered that CMR detects impaired microvascular reperfusion in AMI patients despite successful infarct angioplasty, associated with a lack of recovery of wall motion.

Myocardial perfusion abnormalities

Nidal Al-Saadi investigated first-pass contrast-enhanced CMR to coronary angiography in a clinical setting and refined algorithms for the analysis of signal intensity changes in various setting.

Hb oxygenation changes in stress-induced myocardial ischemia

Matthias Friedrich focused his studies on the further development of using non-contrast CMR techniques to assess tissue Summary oxygenation in acute myocardial ischemia. Using Blood- Oxygen-Level-Dependent magnetic resonance imaging The Cardiovascular Magnetic Resonance (CMR) group at the (BOLD-MRI) to assess tissue oxygenation, he demonstrated Franz-Volhard-Klinik has focused research on the in vivo that adenosine BOLD-MRI detects ischemia in myocardial assessment of functional and structural myocardial abnorma- segments related to severe coronary stenoses. lities related to infective inflammation and coronary heart disease. Future aspects

Early tissue changes in myocardial infarction Matthias Friedrich has accepted a position of an Associate Professor for Cardiology and Director of the Cardiovascular Jeanette Schulz-Menger addresses issues related to myocar- MR Center at University of Calgary, Alberta, Canada. dial hypertrophy and fibrotic scarring related to hypertrophic Andreas Kumar and Hassan Abdel-Aty will join him. Jeanette cardiomyopathy. She was able to show that contrast-enhanced Schulz-Menger will assume responsibility for the group. A magnetic resonance imaging detected infarct-related signal close relationship between Matthias Friedrich and the CMR changes as early as 1 hour after acute myocardial infarction department of the Franz-Volhard-Klinik is being established. (AMI) in humans. Daniel Messroghli is currently spending a two-year stay at the CMR center of the University of Leeds and will return in January 2005 to continue his work (funded by the Marie- Magnetic relaxation properties of myocardial tissue Curie foundation).

Daniel Messroghli focused his research on the development of new approaches to directly measure myocardial magnetic Selected Publications relaxation properties related to ischemia. He demonstrated that T1 mapping visualizes changes in the longitudinal rela- Abdel-Aty H, Zagrosek A, Schulz-Menger J, et al. (2004) xation time induced by acute myocardial infarction. Delayed Enhancement and T2-Weighted Cardiovascular Magnetic Resonance Imaging Differentiate Acute from Chronic Myocardial Infarction. Circulation (in press). Assessment of myocardial water content Taylor AJ, Al-Saadi N, Abdel-Aty H, Schulz-Menger J, Hassan Abdel-Aty investigated myocardial edema related to Messroghli D, Friedrich MG. (2004) Detection of acutely acute myocardial infarction. He performed a study on the impaired microvascular reperfusion following infarct angio- differential aspects of edema in acute and chronic settings and plasty with magnetic resonance imaging. Circulation (in could verify the close correlation of myocardial edema to the press). stage of reperfused infarction. Schulz-Menger J, Gross M, Messroghli D, Uhlich F, Dietz R, Friedrich MG. (2003) Cardiovascular magnetic resonance of acute myocardial infarction at a very early stage. J Am Coll Cardiol 42:513-8. 41

BOLD-MRI images compared to conventional nuclear medicine (SPECT) images to visualize myocardial ischemia. Whereas the SCPECT images reflect the accumulation of Thallium as a surrogate marker, BOLD-MRI directly reflects tissue oxygenation, thus provides molecular information in vivo.

Messroghli DR, Niendorf T, Schulz-Menger J, Dietz R, Friedrich MG. (2003) T1 mapping in patients with acute myocardial infarction. J Cardiovasc Magn Reson 5:353-9.

Friedrich MG, Niendorf T, Schulz-Menger J, Dietz R. (2003) Blood-Oxygen-Level-Dependent Magnetic Resonance Imag- ing in Patients with Stress-Induced Angina. Circulation 108:2219-2223.

Structure of the Group

Group Leader Dr. Matthias Friedrich

Scientists Doctoral Student Dr. Jeanette Schulz-Menger Nico van der Meer Dr. Ralf Wassmuth Dr. Nidal Al-Saadi Study Nurse Dr. Andreas Kumar Melanie Bochmann Dr. Hassan Abdel-Aty (Univ. of Cairo) Dr. Michael Stoeter Dr. Philipp Boye Dr. Anja Zagrosek Dr. Petra Bock Dr. Steffen Bohl Wolfgang Utz 42

Molecular Muscle Physiology ALC-1, while MyHC isoenzymes did not change. Ventricular myosin associated with ALC-1 revealed a higher shortening velocity and rate of force development than normal cross- Ingo L. Morano bridges without ALC-1. Maximal isometric force production per cross-sectional area as well as Ca2+ sensitivity of the force Ca2+ ratio were enhanced. The failing ventricles of patients with dilated cardiomypathy, however, hardly expressed ALC- 1. Therefore, an adenoviral vector containing the human ALC-1 (hALC-1) expression cassete (CASSETTE?) was developed for the upregulation of the hALC-1 in the cardiomyocytes of the failing human heart as a novel gene therapeutic approach.

Regulation of smooth muscle contractility by recruitment of non-muscle myosin in an SM-MyHC knock-out model Smooth muscle cells express three MyHC genes, namely one smooth-muscle-specific (SM-MyHC) as well as two non- muscle-MyHC (NM-MyHCA and NM-MyHCB). We eliminated expression of the SM-MyHC by gene targeting technology. Smooth muscle from knock-out neonatal mice did not exhibit initial phasic contraction while tonic contraction remained normal. Intracellular Ca2+ transients of smooth Contractility of cardiac and smooth muscle is regulated by muscle cells from wild-type and knock-out animals were calcium ions (Ca2+) that enter the cells through voltage-gated similar. Thus, the phasic contraction is generated by L-type Ca2+ channels and subsequently induce the release of SM-MyHC recruitment while the sustained tonic contraction high amounts of Ca2+ from the sarcoplasmic reticulum into state can be produced by NM-MyHC activation. In addition, the myoplasm through calcium release channels (ryanodin both contractile systems in smooth muscle are associated with receptors). Calcium ions activate both intracellular signalling different second messenger pathways Both the SM-MyHC pathways and contraction of the myofibrils. In cardiomyo- and NM-MyHC systems seem to be involved in electro- cytes, they activate the myofibrils by binding to troponin C, mechanical and pharmocomechanical coupling, respectively. which turns the thin filament from an “off” into an “on” state, allowing the molecular motor myosin to interact with the thin filament to produce force and shortening. In smooth muscle Understanding Ca2+-handling proteins cells, Ca2+ form a complex with calmodulin that activates the myosin light chain kinase, an enzyme which phosphorylates a L-type Ca2+ channels are multi-subunit proteins composed of

20kDa regulatory light chain of myosin, thus allowing the the pore-forming a1C subunit (Cav1.2) together with auxi- smooth muscle myosins to generate contraction upon inter- liary subunits, 2/ , and 2. Alterations in the density or action with the thin filaments. Because of their key-roles in function of L-type Ca2+ channels have been implicated in a muscle, we are studying the expression regulation, post-trans- variety of cardiovascular diseases, including atrial fibrilla- lational modifications, and functional roles of the subunits of tion, ventricular hypertrophy, and heart failure. Activation of L-type Ca2+ channel, ryanodine receptor, proteins of the Ca2+ the beta-adrenergic receptor cascade markedly increases Ca2+ signalling pathways, and type II myosin in cardiac and influx via protein kinase A (PKA)-dependent phosphorylation smooth muscle. Any change in these key proteins, by muta- of the channel subunits: 1C, 2 or still unrecognized association, differential gene expression, alternative splicing of the ted proteins. In an attempt to define the molecular details of transcripts, or post-translational modification modulates channel phosphorylation, we have identified the 700-kDa cardiac and smooth muscle function. Understanding muscle ahnak as tightly associated protein and prominent PKA target contraction regulation at the molecular and functional level provides an opportunity to develop new therapies for the treatment of cardiac and smooth muscle dysfunction.

Confocal images depicting the localization of ahnak-C2 in the human heart. Longitudinal (A) and cross (B) sections of human myocardium were stained for ahnak- Understanding the molecular motor C2 (green) and nuclei (red). Ahnak-C2 labels the T-tubular system (small arrows), the surface sarcolemma (star), and the intercalated disks (big arrow) (C) High magnification of a transversal section showing one myocyte. The T-tubular system (arrows) is oriented Essential myosin light chain isoforms regulate human heart radially inside the myocyte (from: Hohaus et al. 2002, FASEB 16: 1205–1216). contractility Type II myosin isoenzymes are hexamers of about 500 kDa AB C composed of two heavy chains (MyHC) and 4 light chains (MLC). Atrium- and ventricle-specific essential (ALC-1 and VLC-1, respectively) and regulatory (ALC-2 and VLC-2, respectively) MLC exist in the human heart. Cardiomyocytes of hypertrophied ventricles of patients with congenital heart diseases and hypertophic cardiomyopathy reexpressed 43

in mammalian cardiomyocytes. Next, we characterized ahnak Graduate and Undergraduate Students in normal human myocardium as a peripheral membrane Katarina Wetzel protein associated with the cytoplasmic aspect of the plasma Radu Iliescu membrane including T-tubular structures (Fig. 1). Using Valéria Lamounier-Zepter truncated ahnak fragments, we demonstrated the presence of Christiane Woischwill multiple 2-subunit interaction sites within ahnak’s carboxy- Daria Petzhold terminal. This ahnak domain was also defined to be respon- Ihab Abdelaziz sible for F-actin binding. Together, localization and interac- Gisela Dobernack tion partner suggest a role of cardiac ahnak as sarcolemma support protein and as a linker between Ca2+ channels and Technical Assistants subsarcolemmal cytoskeleton. Recent electrophysiological Dr. Monika Kott experiments demonstrated for the first time that carboxy- Petra Pierschalek terminal ahnak fragments modulate specific aspects of Ca2+ Steffen Lutter channel gating (ICaL) properties such as an increase in ICaL Wolfgang-Peter Schlegel amplitude and a slowing of inactivation. Hence, our results Katrin Jäger suggest that binding of the 2-subunit to the subsarcolemmal giant ahnak protein reprimes the 1C- 2-subunit interaction Secretariat and the relief of this inhibition increased the Ca2+ inward Manuela Kaada current. Ongoing studies include the generation of an ahnak- knock-out mouse model and a screening program to identify ahnak mutations in patients suffering from cardiomyopathies.

Selected Publications

Lofgren M, Ekblad E, Morano I, Arner A (2003) Non-muscle myosin motor of smooth muscle. J. Gen. Physiol. 12: 301– 310

Hohaus A, Person V, Behlke J, Schaper J, Morano I, Haase H (2002) The carboxyl-terminal region of ahnal provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton. FASEB 16: 1205–1216

Morano I., Chai G.-X., Baltas L. G., Lamounier-Zepter V., Kott M., Haase H., Walther T. and Bader M. (2000). Smooth muscle contraction without smooth muscle myosin. Nature Cell Biology, 2, 371–375

Morano I. (1999). Tuning the human heart molecular motors by myosin light chains. J. Mol. Med. 77, 544–555

Haase H., Podzuweit T., Lutsch G., Hohaus G., Kostka S., Lindschau C., Kott M., Kraft R. and Morano I. (1999) Signa- ling from b-adrenoceptor to L-type calcium channel: identification of a novel cardiac protein kinase A target possessing similiarities to ahnak. FASEB J. 13, 2161–2172

Structure of the Group

Group Leader Prof. Dr. Ingo Morano

Scientists Dr. Hannelore Haase Dr. Peter Karczewski Yana Khalina (Guest) 44

Cell Polarity During Development Large-scale screens for embryonic lethal mutations in zebra- of Drosophila and Zebrafish fish have isolated several mutations that affect epithelial integrity. One of these mutations, heart and soul (aPKC), affects early development and the formation of several polari- Salim Abdelilah-Seyfried zed epithelia. Consistent with a conserved role of zebrafish aPKC, the protein is required for the formation and maintenance of adherens junctions in the polarized epithelia of the retina, neural tube, and digestive tract. During early stages of organogenesis, heart and soul appears to regulate the apical clustering and maintenance of adherens junctions. In addition to the epithelial defects, heart and soul affects the morpho- logies of the heart tube and the gut and some of its associated organs.

We have performed a functional analysis of aPKC by using a combination of antisense oligonucleotide morpholinos mediated gene “knock down” and coexpression of mutant aPKC mRNAs. This approach provides conclusive evidence that activity of the catalytic domain is essential in the context of vertebrate cell polarity and organ morphogenesis. More- over, it identifies several highly conserved residues that have been implied in potential regulatory roles and demonstrates Summary that they are indeed critical for aPKC function.

Epithelial cells polarize along their apico-basal axis and sepa- Research in our laboratory is currently directed towards iden- rate apical from basolateral membrane compartments during tifying and characterizing the direct downstream targets of development. Mature epithelial cells are highly polarized aPKC in the context of cell polarity and organ morphogene- with separate apical and basolateral membrane compartments, sis. Furthermore, we are involved in the cloning and charac- each with a unique composition of lipids and proteins. Within terization of other zebrafish mutations that affect cellular mature epithelial tissues, cell polarity regulates cellular mor- polarity and epithelial integrity. The identification of the phology, intracellular signaling, asymmetric cell division, cell molecular pathways involved in vertebrate epithelial morpho- migration, cellular and tissue physiology as well as complex genesis may lead to relevant animal models for human organ morphogenesis. We are interested in the molecular epithelial pathologies and allow for the development of novel mechanisms that regulate the polarization of epithelial cells therapeutic approaches. and are using zebrafish and fruitfly (Drosophila) as our experimental systems. We would like to answer the following questions: How do the different protein complexes that Bazooka in cell migration establish cell polarity interact with each other? What are the signals by which cell polarity is mediated within cells? How During the development of multicellular organisms, various is cell polarity regulated within epithelial sheets during types of directed cell migration occur that contribute to the morphogenesis of tissues and organs? Our long-term interest development of different tissues. These include the migration is to understand how the cellular mechanisms controlling cell of neural crest cells, hematopoietic stem cells, and germ cells. polarity shape our own bodies. Gaining a better understanding of the mechanisms that govern normal cell motility and invasion is crucial for understanding development and may also contribute to understanding forms Cell polarity in zebrafish epithelial formation and of aberrant cell invasion and migration of metastatic tumor organogenesis cells.

The establishment and maintenance of polarity is an essential Border cell migration during Drosophila oogenesis is one feature of eukaryotic cells. At the core of initiating and main- well-studied example of invasive and directed migration. taining cellular polarity is the conserved Par protein complex Border cells are specified within the anterior follicular epithe- that contains an atypical protein kinase C (aPKC) and the lium that surrounds the germ cells in each egg chamber, dela- PDZ domain containing Par6 protein. The role of this protein minate from the monolayer epithelium and, in a highly complex in epithelial formation is best understood in Droso- stereotyped fashion, invade the germ cell cluster. First, they phila where Bazooka (Drosophila Par-3), Par-6, and aPKC undergo directed cell migration towards the oocyte and then localize to the apico-lateral membrane of embryonic epi- turn dorsally. thelia, just apical and partially overlapping Armadillo (-catenin) localization at the zonula adherens. The disrup- We showed that wild-type bazooka (the Drosophila homolog tion of normal gene function causes epithelial defects, includ- of par-3) is required during cell invasion of epithelial follicle ing loss of cellular polarity, loss of the zonula adherens, and cells mutant for the tumor suppressor discs large. Clonal changes in cell shape. studies indicate that follicle cell Bazooka is a permissive factor during cell invasion, possibly by stabilizing adhesion 45

Structure of the Group

Group Leader Salim Abdelilah-Seyfried

Scientists Dr. Nana Bit-Avragim* Dr. Eva Hartfuss*

Graduate Students Elena Cibrian* David Hava* Sabine Seipold Heart morphogenesis defects in zebrafish epithelial mutant. (A) The zebrafish wild-type Stefan Rohr embryonic heart at 30 hours of development visualized with cardiac myosin light chain 2 probe. The heart is an elongated two-chambered tube with an anterior atrium (a) and posterior ventricle (v). (B) In comparison, the heart tube is not elongated in nagie oko Technical Assistants mutant embryos. Petra Heere Jacqueline Klewer*

* part of the period reported

between the invading somatic cells and their substrate, the germ line cells. Genetic epistasis experiments demonstrate that bazooka acts downstream of discs large in tumor cell invasion. In contrast, during the migration of border cells, Bazooka function is dispensable for cell invasion and motility, yet is required cell-autonomously in mediating cell adhesion within the migrating border cell cluster. Taken together, these studies reveal that Bazooka functions distinctly in different types of invasive behaviors of epithelial follicle cells, potentially by regulating adhesion between follicle cells or between follicle cells and their germ line substrate.

Selected Publications

Abdelilah-Seyfried, S., Cox, D.N. and Jan, Y.N. (2003). Bazooka is a permissive factor for the invasive behavior of discs large tumor cells in Drosophila ovarian follicular epithelia. Development 130, 1927-1935.

Hauptmann G, Belting HG, Wolke U, Lunde K, Soll I, Abde- lilah-Seyfried S, Prince V, Driever W. (2002). spiel ohne grenzen/pou2 is required for zebrafish hindbrain segmentation. Development 129, 1645-1655.

Horne-Badovinac S, Lin D, Waldron S, Schwarz M, Mbamalu G, Pawson T, Jan Y, Stainier DY, Abdelilah-Seyfried S. (2001). Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis. Current Biology 11, 1492-1502.

Belting HG, Hauptmann G, Meyer D, Abdelilah-Seyfried S, Chitnis A, Eschbach C, Soll I, Thisse C, Thisse B, Artinger KB, Lunde K, Driever W. (2001). spiel ohne grenzen/pou2 is required during establishment of the zebrafish midbrain-hindbrain boundary organizer. Development 128, 4165-4176.

Cox, D.N., Abdelilah-Seyfried, S., Jan, L.Y. and Jan, Y.N. (2001). Bazooka and atypical protein kinase C are required to regulate oocyte differentiation in the Drosophila ovary. PNAS 98, 14475-14480. 46

Cell Biology of Cardiovascular picture of the nucleus with many discrete and distinguishable Diseases subnuclear compartments involved in DNA or RNA metabolism. We are studying the coordination of the multiple enzymatic activities involved in the replication of the genome M. Cristina Cardoso at every cell division cycle. (in collaboration with Heinrich Leonhardt) To study the dynamic regulation of these nuclear structures during the cell cycle in vivo and in real time, we have established an approach for the visualization of DNA replication in living cells using translational fusions of different replications factors to green (GFP) or red (DsRed) fluorescent proteins. Using high resolution time lapse microscopy, we could show that replication site patterns within the nucleus change in a characteristic manner throughout S phase.

To investigate whether the replication factors remain stably bound at replication foci or whether they are in constant exchange, we have used biochemical in situ extractions as well as fluorescence photobleaching techniques. Both experimental approaches showed that the PCNA (proliferating cell nuclear antigen) clamp was tightly bound at replication sites, showing only little exchange, if any. A comparison with Differentiation and proliferation of muscle cells another replication factor RPA (single-stranded DNA-binding protein) involved in the initiation of DNA replication showed During terminal differentiation, striated muscle cells perma- that, while RPA exchanged in a time frame of seconds, PCNA nently withdraw from the cell cycle and become refractile to showed virtually no turnover within several minutes. This has growth stimulation. We are interested in the molecular mechanisms regulating the establishment and maintenance of terminal differentiation and in devising ways to transiently reverse this state to achieve tissue regeneration. We have pre- Figure 1 viously shown that this proliferation arrest is an actively Propagation of DNA replication The image shows the progression of DNA replication in live mammalian cells. Spatio- maintained process that can be reversed upon transgenic temporal changes of DNA replication were followed with a GFP-PCNA fusion protein by expression of the simian virus 40 large T antigen (SV40 TAg). time lapse microscopy. This time overlay shows the distribution of replication sites in To avoid the hazards of gene therapy-based strategies, we are green and their redistribution 15 minutes later in red. New replication sites (red) are de novo assembled at adjacent sites with replication proteins from the nucleoplasmic pool. developing approaches to directly deliver the gene products “Reprinted from Molecular Cell, Vol. 10, Sporbert et al., 1355-1365, Copyright (2002) (i.e., the proteins) to these cells. Taking advantage of the with permission from Elsevier”. intercellular trafficking properties of the herpes simplex virus I VP22 protein, we have directly delivered SV40 TAg to striated muscle cells via fusion with VP22 and shown that it can stimulate cell proliferation. This protein transduction method allows for the simultaneous delivery of mixtures of regulatory proteins in a dose- and time-controlled fashion and it is easy to combine with the application of other compounds. We are presently optimizing this technology for tissue regeneration in vivo and for the expansion of differentiated stem cells (embryonic and adult) in vitro. In addition, in collaboration with the group of R. Kettritz (FVK), we are testing the applicability of this approach to other terminal differentiated cells, such as human neutrophils, which play an important role in the process of vasculitis.

Nuclear organization and genome replication

Although the nucleus is the hallmark of eukaryotic cells, we still know remarkably little about its structure and function. For a long time, the nucleus has been underestimated as a mere repository of the genetic information packed into chromatin, freely floating like noodles in a soup of amorphous nucleoplasm. However, in the last decades the development of antibodies to nuclear components combined with the ability to fluorescently tag proteins has revealed a different 47

lies the ordered activation of later replication origins and sets the replication program.

Translation and replication of epigenetic information

Together with the genetic information, the epigenetic information is also duplicated and maintained over many cell generations. One of the essential epigenetic modifications in mammalian genomes is the methylation at position 5 of cyto- sines residues. We are analyzing different proteins involved in the maintenance and change of this epigenetic modification and their dynamic interaction with the replication machinery. Both the replication of genetic and epigenetic information are required for stable gene expression patterns.

We are approaching these questions via the identification and characterization of functional domains of the known DNA methyltransferases (Dnmt1, 2, 3a and 3b) and methyl-cytosine binding proteins (MeCP2, MBD1-4). We have recently found that Dnmt1 binds to the replication machinery during S Figure 2 Genome replication program. phase via its interaction with PCNA and remains bound to Mammalian cells expressing a GFP tagged DNA ligase I were pulse labeled for 10 minu- centromeric heterochromatin during both G2 phase and M tes with the thymidine analog bromodeoxyuridine, followed by chase with 10-fold excess of thymidine. Three hours later, the cells were fixed and the incorporated phase via a separate targeting sequence. Deletion of this nucleotide detected with specific antibodies (red) together with simultaneous detection sequence as well as its overexpression indicate an essential of the GFP-DNA ligase I (green) localization. The image shows a projection of a z-stack role in the methylation of centromeric repeat sequences and in of confocal images. The early replicating chromatin (labeled with the nucleotide) consisting mostly of euchro- chromatin stability and organization. matic regions is shown together with the late replicating chromatin (labeled three hours later with the tagged replication protein) comprising mostly constitutive heterochromatic centers. This spatial and temporal program of replication is followed at every cell cycle. Mice carrying a hypomorphic Dnmt1 allele, which reduces Dnmt1 expression to 10% of wild type levels, exhibited genomic hypomethylation and developed aggressive tumors with a high incidence of chromosome 15 trisomy. These results pro- lead us to propose an alternative model for DNA replication, vide a causal link between DNA hypomethylation and tumor whereby the PCNA clamp stays bound throughout the synthe- formation, possibly by promoting genomic instability. sis of several Okazaki fragments. This could be achieved, as it was suggested for the DNA polymerase, by coupling the We are now investigating the role of other functional domains leading and lagging strand PCNA-polymerase complex to- in vivo using transgenic animal approaches as well as testing gether. We are currently testing this model by simultaneously the role in the maintenance of other epigenetic modifications. measuring the on/off rate of PCNA and PCNA-binding repli- We are also investigating whether these functions are con- cation factors in living cells. Since these fluorescence imag- served during evolution. Furthermore, we are studying the ing techniques are based on averages of thousands of molecu- role of methyl-cytosine binding proteins in the translation of les, we, in collaboration with the groups of U. Kubitscheck epigenetic information. and H. Leonhardt, are now tracing single molecules within living cell nuclei. Selected Publications Replication of the mammalian genome starts at tens of thousands of origins that are activated at specific times during Gaudet, F., Rideout, W. M. 3rd, Meissner, A. Dausman, J., S phase raising the question of how this replication program Leonhardt, H. and Jaenisch, R. (2004). Maintenance of IAP is coordinated. Importantly, the spatio-temporal progression methylation by Dnmt1 in pre and postimplantation embryo- of DNA replication is inherited through consecutive cell genesis Mol. Cell. Biol., 24: 1640-1648. division cycles. Our fluorescence photobleaching analyses showed that the transition from earlier to later replicons Choi, M., Rolle, S., Wellner, M., Cardoso, M. C., Scheidereit, occurs by disassembly into a nucleoplasmic pool of rapidly C., Luft, F. C. and Kettritz, R. (2003). Inhibition of NF-B by diffusing subcomponents and reassembly at newly activated TAT-NEMO-binding domain peptide accelerates constitutive sites. A careful examination of the temporal and spatial apoptosis and abrogates LPS-delayed neutrophil apoptosis. assembly of new PCNA molecules by overlaying the images Blood, 102: 2259-2267. collected at consecutive times indicated that PCNA assembled in non-overlapping sites. These replication sites were in Gaudet, F., Hodgson, J. G., Eden, A., Jackson-Grusby, L., close proximity to earlier ones suggesting that activation of Dausman, J., Gray, J. W., Leonhardt, H. and Jaenisch, R. neighboring origins may occur by a domino effect possibly (2003). Induction of Tumors in Mice by Genomic Hypo- involving local changes in chromatin structure and accessi- methylation. Science, 300: 489-492. bility. We are now trying to dissect the mechanism that under- 48

Derer, W., Easwaran, H. P., Leonhardt, H., and Cardoso, M. C. (2002). A novel approach to induce cell cycle reentry in terminally differentiated muscle cells. The FASEB J. 16: 132- 133.

Sporbert, A., Gahl, A., Ankerhold, R., Leonhardt, H. and Cardoso, M. C. (2002). DNA polymerase clamp shows little turnover at established replication sites but sequential de novo assembly at adjacent origin clusters. Mol. Cell, 10: 1355-1365.

Structure of the Group

Group Leader Dr. M. Cristina Cardoso

Scientists Dr. Heinrich Leonhardt (collaborator) Dr. Jean B. Margot* Dr. Anje Sporbert Dr. Hariharan P. Easwaran Dr. Hideki Sakamoto*

Graduate and Undergraduate Students Francois Gaudet Robert Martin* Gilla Tünnemann* Danny Nowak Maik Grohmann* David Grünwald* Antje Krella*

Technical Assistants Anja Gahl Ingrid Grunewald Petra Domaing Marion Bengs*

* part of the time reported. 49

Immunology of Cardiovascular renoceptor and the improvement of heart function support the Diseases hypothesis that the anti- 1-adrenoceptor antibodies may play a role in the pathophysiology of myocarditis and DCM.

Gerd Wallukat To confirm this hypothesis, we developed a specific immunoadsorption column. Based on our epitope analysis, a peptide column was generated that selectively removes the anti- 1- adrenoceptor autoantibodies. It was shown in a pilot study that the treatment of DCM patients with this specific adsorption results in improved cardiac function.

Autoantibodies in hypertension

Furthermore, we have investigated the role of autoantibodies in essential and therapy refractory hypertension. In the sera of patients with this disease, we detected autoantibodies directed against the 1-adrenoceptor. These autoantibodies recognize epitopes on the first or second extracellular loop of the 1-adrenergic receptor and act like 1-adrenergic agonists. In patients with hypertension refractory to therapy, more than 80% of the patients were antibody positive. In patients with The interest of our group is focussed on immunological pro- malignant hypertension, those with acute vascular kidney cesses in cardiovascular diseases. In several cardiovascular rejection, and those with preeclampsia, we observed autoanti- diseases, we discovered functional autoantibodies against bodies against the angiotensin II AT1-receptor. In preeclamp- extracellular structures of G-protein coupled receptors.We tic patients, this antibody is detectable after the 20th week of observed autoantibodies against adrenergic receptors and pregnancy and disappears after delivery. These agonist-like

AT 1-receptors in the sera of patients with myocarditis, dilated anti-AT1-receptor antibodies induce formation of the trans- cardiomyopathy (DCM), and hypertension. These autoantibo- cription factors AP-1 and NFB and activate NADPH dies recognize epitopes on the first or second extracellular oxidase. These functional autoantibodies are found in more loop of the receptors and act like the corresponding pharma- than 90% of preeclamptic women investigated and may play cological agonists. In patients with myocarditis and dilated a role in elevating vascular resistance and promoting hyper- cardiomyopathy, but also in Chagas’ disease, the autoanti- tension and cardiac hypertrophy in these patients. bodies recognize the 1-adrenoceptor and, in some patients, the muscarinic M2 receptor as well. In recent years, we have investigated in more detail the effects of these autoantibodies. Autoantibodies in Raynaud’s syndrome We believe that the antibodies stabilize the agonistic confir- mation of the receptors resulting in the agonist-like effect. Raynaud’s syndrome is characterized by a cold induced reduction of the blood flow into small vessels of the extremi- ties. The pathogenesis of this disease is not fully understood. Autoantibodies in myocarditis and dilated We have observed that the sera of patients with Raynaud’s cardiomyopathy (DCM) syndrome contain functional autoantibodies against protease- activated receptors (PAR). The antibodies react with the The suggestion that the anti- 1-adrenoceptor autoantibody second extracellular loop of both the PAR-1 (thrombin recep- may play a role in the pathogenesis of DCM is supported by tor) and the PAR-2 (tryptase receptor) because the epitope of similar findings in patients with myocarditis, a disease widely the antibodies on the receptor is identical for these receptor held to be a precursor of DCM. We observed that in patients subtypes. Our investigations aim to elucidate the role of these with these autoantibodies disappeared during the process of autoantibodies in the development and/or maintenance of this healing. In parallel the left ventricular ejection fraction and disease. heart rate were normalized. Moreover, it was shown that immunization of animals with the 1-adrenoceptor or with peptides corresponding to this receptor caused a disease that Selected Publications corresponded to dilated cardiomyopathy. Dechend, R., Viedt, C., Müller, D.N., Ugele, B., Brandes, Based on our autoimmune hypothesis, we proposed new R.P., Wallukat, G., Park, J.K., Theuer,J., Fiebler, A., Homuth, therapeutic possibilities to treat patients with endstage dilated V., Dietz, R., Haller, H., Kreuzer, J., and Luft, F.C. (2003) cardiomyopathy. One is unspecific immunoadsorption using AT1-receptor agonistic antibodies from preeclamptic patients columns that remove all IgG immunoglobulins from the stimulate NADPH oxidase. Circulation 107, 1632-39. patient’s plasma. After this treatment, a marked improvement in cardiac function and normalization of the cardiac size were observed. The strong correlation observed between the reduction in the number of circulating autoantibodies to the 1-ad- 50

Wallukat, G., Neichel, D., Nissen, E., Homuth, V., and Luft, F.C. (2003) Agonistic autoantibodies directed against the angiotensin II AT1 receptor in patients with preeclampsia. Can J Physiol Pharmacol 81, 79-83.

Dörffel, Y., Wallukat, G., Bochnig, N., Homuth, V., Herberg, M., Dörffel, W., Pruss, A., Chaoui, R., and Scholze, J. (2003) Agonistic AT1 receptor autoantibodies and monocyte stimulation in hypertensive patients. Am J Hyperten 16, 827-33.

Wallukat, G., Müller, J., and Hetzer, R. (2002) Specific removal of beta1-adrenergic autoantibodies from patients with idiopathic dilated cardiomyopathy. NEJM 347, 1806.

Structure of the Group

Group Leader Dr. Gerd Wallukat

Scientists Dr. Sabine Bartel Dr. Wolfgang Schulze

Graduate and Undergraduate Students Janette Freier Fu Qin Marion Janczikowski

Technical Assistants Karin Karczewski Monika Wegener

Secretariat Dana Lafuente 51

Neuromuscular and Cardiovascular Stretch signal Cell Biology Titin is a unique molecule that contains elastic spring elements and a kinase domain, as well as phosphorylation Michael Gotthardt sites. Therefore, it has been frequently speculated that titin and invertebrate giant titin-like molecules could act as a stretch sensor in muscle. More recently, this concept has been supported by studies on human dilative cardiomyopathies which suggest an impaired interaction of titin with its regulatory ligands Tcap/telethonin and MLP protein. However, so far it has remained unknown how the stretch signal is processed, i.e. how the mechanical stimulus stretch is converted into a biochemical signal.

To understand the stretch signaling pathway, we utilize mouse genetics, biomechanics, and signal transduction analysis to study the interplay of titin’s elastic and catalytic regions and their regulation in a stretch-dependent fashion.

Smooth muscle and non-muscle titins

Introduction Only recently, the muscle protein titin has been proposed to perform non-muscle functions, following its localization to Titin is a protein with multiple elastic and signaling functions various cell compartments such as the chromosomes of droso- derived from a complex subdomain structure (see also pro- phila neuroblasts and the brush border of intestinal epithelial gress report by Ludwig Thierfelder). In striated muscle, titin cells. Titin has been implicated in cytokinesis through locali- forms a continous filament system and serves as a template to zation to stress fibers/cleavage furrows and in chromosome assemble the sarcomere providing multiple binding sites as condensation through localization to mitotic chromosomes. depicted below. Drosophila melanogaster deficient in the titin homologue D-titin show chromosome undercondensation, premature Titin is relevant in human disease, not only for its role in late sister chromatid separation, and aneuploidity. stage cardiomyopathies, where changes in titin-isoform expression impair tissue elasticity, but also as the primary Our preliminary data indicate that titin is present in virtually defect in various cardiac as well as skeletal muscle diseases. every cell-type tested. Nevertheless, our knockout of titin’s Currently, it is not known why the titin mutations identified M-line exon 1 and 2 does not show an obvious non-muscle so far cause either cardiac diseases or skeletal muscular phenotype, such as a defect in implantation or in cell-migra- dystrophy, although the mutated titin segments are expressed tion. Accordingly, we have extended the analysis of our titin in all striated muscles. knockout animals to actin-filament dependent functions (cytokinesis and chromosome segregation) to establish the Recent work has shown that even smooth muscle and non- role of titin in non-muscle cells. muscle cells express titin or titin-like proteins, albeit at low levels compared to striated muscle. A plethora of non-muscle functions have been proposed for titin such as a role in Functional analysis of individual titin domains chromosome condensation, chromosome segregation, or in the assembly of actin stress fibers but none of these has been To lay the groundwork for the in vivo analysis of titin’s multi- demonstrated conclusively. So far, there is no comprehensive ple signaling, elastic, and adaptor domains and their interplay investigation of non-muscle and smooth muscle titin expres- in muscle as well as in non-muscle cells, we have started with sion or splicing and conflicting data on its proposed function. the generation of various titin mutant mice (knock-in and conditional knockout animals) and established a tissue culture Our long-term goal is to establish the role of titin in muscle system to study titin’s muscle and non-muscle functions. We and non-muscle function and disease, with the main emphasis utilize a combination of cell-biological, biochemical, and on signal transduction and biomechanics. We have estab- genetic tools to establish titin as a stretch sensor converting lished the structure of the mouse titin gene and used gene mechanical into biochemical signals in muscle and in non- targeting to create a set of knockout mice, which can be in- muscle cells. duced to express various mutant titin molecules lacking critical subdomains. With these mice, we have demonstrated Understanding structural and biomechanical as well as signal- that titin is crucial for embryonic development, assembly of ing and metabolic functions of titin will help elucidate the the sarcomere, and muscle function. pathomechanism of various cardiovascular diseases and cancer and ultimately aid the development of suitable therapeutic strategies. 52

Schematic diagram of the sarcomere (modified from Gregorio et al., Curr. Opin. Cell. Biol. 11: 18-25, 1999). Titin forms a continuous filament system along the muscle fiber in vertebrate striated muscle overlapping in the M-line (titin C-terminus) and in the Z-disc (N-terminus). The titin kinase is found near the edge of the M-line region, while the elastic PEVK resides in the I-band. Titin interacts with a plethora of sarcomeric proteins, such as T-cap and C-protein.

Selected Publications Structure of the Group

Boucher, P.#, Gotthardt, M. #, Li, W.P., Anderson, R.G., Herz, Group Leader J. (2003). LRP: Role in Vascular Wall Integrity and Protection Dr. Michael Gotthardt* from Atherosclerosis. Science 300, 329-32 (# = equal contribution). Scientists Dr. Anette Hohaus* Gotthardt, M., Hammer, H.E., Hübner, N., Monti, J., Witt, C.C., McNabb, M.M., Richardson, J., Granzier, H., Labeit, S., Graduate and Undergraduate Students Herz, J. (2003). Conditional expression of mutant M-line Michael Radke* titins results in cardiomyopathy with altered sarcomere Katy Raddatz* structure. J. Biol. Chem. 278, 6059-65 Steffi Weinert* Nora Bergmann* Holtwick, R., Gotthardt, M., Skryabin, B., Steinmetz, M., Potthast, R., Zetsche, B., Hammer, R.E., Herz, J., and Kuhn, Technical Assistants M. (2002). Smooth muscle-selective deletion of guanylyl Beate Goldbrich* cyclase-A prevents the acute but not chronic effects of ANP Katrin Räbel** on blood pressure. PNAS 99, 7142-7147. * part of the period reported Vasandani, C., Kafrouni, A.I., Caronna, A., Bashmakov, Y., ** guest, part of the period reported Gotthardt, M., Horton, J.D., and Spady, D.K. (2002). Upregu- lation of hepatic LDL transport by n-3 fatty acids in LDL receptor knockout mice. J Lipid Res 43, 772-784.

Boucher, P., Liu, P., Gotthardt, M., Hiesberger, T., Anderson, R.G.W., and Herz, J. (2002). Platelet-derived Growth Factor Mediates Tyrosine Phosphorylation of the Cytoplasmic Domain of the Low Density Lipoprotein Receptor-related Protein in Caveolae. J.Biol.Chem. 277, 15507-15513.

Bang, M.L., Centner, T., Fornoff, F., Geach, A.J., Gotthardt, M., McNabb, M., Witt, C.C., Labeit, D., Gregorio, C.C., Granzier, H., and Labeit, S. (2001). The complete gene sequence of titin, expression of an unusual approximately 700-kDa titin isoform, and its interaction with obscurin identify a novel Z-line to I-band linking system. Circ Res 89, 1065-1072. Metabolic Diseases, Genetics, Genomics, and Bioinformatics

Bioinformatics score of linkage disequilibrium, LD) varied with their distance on the genome. The full genomic structure of all loci could be established for all gene loci by reference to the NCBI data Jens Reich base of the human genome. About one third of the SNPs were (in collaboration with Peer Bork) found in the coding region (non-synonymous and synonymous), one-third in the intron segments, and one-third in non-translated extragenic regions (promoters etc.). We achieved a full coverage of the genomic loci by around 12 common SNPs. All gene loci were thus represented by considerable, statistically highly significant linkage disequilibrium (LD). Individual variation of the human genome and its role in lipid metabolism: genetic-epidemiological These data are in agreement with population-genetic models study of risk factors for arteriosclerosis for a fully outbred panmictic population in Germany and also with published data on other genome sections. Such a situa- The lipid network group (J.R.) continued its participation in a tion allows the LD mapping of functional gene alleles at small joint study to evaluate the importance of single nucleotide distances from the marker SNP. To this end, we established polymorphisms (SNPs) and splicing variants in the human the SNP-haplotype structure of all loci by way of analysis of genome. This was combined with an application study invol- the family pedigrees of all subjects, since they were recruited ving 1500 healthy individuals recruited by Friedrich Luft at as nuclear families in a systematic field working project. the Franz-Volhard-Clinic (FVK) in which 93 SNP variants at Haplotyping was achieved with a newly developed computer 13 relevant gene loci were measured together with 5 clinically program (Rohde & Fürst, 2001) that permits, with high confi- important indicators of human lipoprotein metabolism. This dence, the establishmentof the chromosomal phase of SNP is the first time that the individual genotype of common SNPs positions when diploid genotype data from nuclear families (i.e. > 3% population frequency in a German population are available, as was our case. It was found that, in all of the sample) relevant to a metabolic pathway were correlated with studied gene loci, only a few (4to 5) haplotypes accounted for the physiological level of the resultant phenotype. the genotype of about 80-90% of the population sample. This means that chromosomal haplotypes of common SNPs are Lipid traits are the major risk factor for arteriosclerosis and its very “old”genomic structures (i.e. many tens of thousands severe complications (e.g., myocardial infarction, stroke, generations old, not dissolved in the whole population by etc.). Physiologically valid phenotypic values (plasma levels extensive recombination) and may therefore be reliable mar- of total cholesterol, TC; triglyceride, TG; low density lipo- kers of functional alleles that explain the variation and risk protein, LDL, high density lipoprotein, HDL; and the impor- status of the phenotype. This warrants a genotype-phenotype tant clinical risk factor LDL/HDL) were measured at the FVK association study if controlled for stratification into subpopu- under standardized conditions and SNP genotyping was done lations. by Peter Nürnberg´s MDC Genomic Mapping Centre. Earlier comparative studies on homozygotic and dizygotic twins The association was evaluated in combination with linkage (Busjahn & Luft) had established that a global heritability tests according to modern models of biometric genetics, component of between 30 and 40% contributes to the indi- which partition the total variance into additive genetic, poly- vidual lipid level in humans. As SNPs are the most common genic, and environmental components. Comparison of geno- genetic variants, we wanted to learn which of them change the type-phenotype correlation between and within families phenotype levels to a statistically significant extent. This enabled a control for stratification (sample inhomogeneity) genotype-phenotype correlation was to be integrated into a effects. pathway model that we have developed on the basis of theo- retical studies of metabolic models (H. Knoblauch et al., We addressed the following questions: 2000). The data obtained were subjected to an exhaustive • What is the degree of heritability of lipid traits in our mathematical-statistical analysis using advanced computer sample ? techniques. • What part of the genetic component is due to the candidate loci which we genotyped, and which complementary part The studied polymorphisms showed common variation in the is due to gene loci not measured? sample. The allelic association of these SNPs (expressed as 54

• What is the contribution of individual candidate loci to the In parallel with these studies, we investigated the importance whole genetic variation ? of splicing variants in the human genome, based on genomic EST date bases, with special emphasis on cancer tissues. We This analysis was done in the following stepwise way: assembled a splice site database which has been published in Comparing general variance and interfamilial covariance of the internet and in peer-reviewed papers (H. Pospisil). The the lipid values, we established that 38% of the variation of possible importance of splice variants as markers or prog- HDL and 26% of the variation of LDL could be explained as nostic predictors of colon cancer was studied in collaboration inherited (heritability estimate). Introduction of the SNP with the Robert-Rössle-Klinik (P. Schlag, W. Kemmner). genotypes and/or SNP haplotypes into the analysis permitted the separation of the relative contribution of our genotyped The haplotype study group (K. Rohde) developed an entropy loci as compared to the polygenic background. In looking at measure for linkage disequilibrium over multilocus haplotype HDL and LDL, about 50% and 75% of the genetic variation blocks (M. Nothnagel) and studied the association of genetic factor, respectively, was attributed to our candidate gene loci. traits to SNP haplotypes using an EM algorithm with Markov Thus, we could show that a major part of the genetic influence Chain Monte Carlo Techniques. can be accounted for by our measured genotypes.

We devised a variance partition method that could estimate Selected Publications the contribution of individual gene variation to the phenotype variation. The figure shows the result for HDL and LDL. In Rohde, K. and Fürst, R. (2003). Association of Genetic Traits the case of HDL, the loci of hepatic lipase, CETP and ABCA1 to Estimated Haplotypes from SNP Genotypes Using EM are the main contributors, whereas ApoE, CETP, and hepatic Algorithm and Markov Chain Monte Carlo Techniques. lipase are the main factors responsible for the variation in the Human Heredity 56, 41-47. LDL phenotype. Knoblauch, H., Bauerfeind, A., Krähenbühl, Ch., Daury, A., The results (submitted) are a “proof of principle” of the “com- Rohde, K., Bejanin, St., Esseoux, L., Schuster, H., Luft, F.C. mon-variant-explains-common-trait”-hypothesis (Collins & and Reich, J.G. (2002). Common haplotypes in five genes Chakravarti) for complex pathways. This is of high relevance influence genetic variance of LDL and HDS cholesterol in the for the prediction of the genetic contribution to the risk status general population. Human Molecular Genetics 11, 1477- of individuals. At present, we are exploring the possible ap- 1485. plication of the results for SNP diagnostics in lipid-relevant genes, with special emphasis on gender and age factors. There Brett, D., Pospisil, H., Valcárcel, J., Reich, J. and Bork, P. are a considerable number of haplotypes that allow a predic- (2002). Alternative splicing and genome complexity. Nature tion of the risk or protection status of subjects with apparently Genetics 30, 29-30. normal cholesterol values with advancing age.

Locuswise contribution of lipid-relevant gene loci to the genetic variance of LDL and HDL variance in the German population. Results are obtained from a multiple association analysis considering SNP-haplotypes. Panel a: LDL; Panel b: HDL. (APOE - apoprotein E; APOB – apoprotein B; LDLR – LDL receptor; LIPC – hepatic lipase; CETP – cholesteryl ester transfer protein; ABCA1 – ABC transporter A1; LPL – lipoprotein lipase)

Fig 1. a) LDL-cholesterol Fig 1. b) HDL-cholesterol 55

Bauerfeind, A., Knoblauch, H., Schuster, H., Luft, F. C. and Reich, J. G. (2002). Single nucleotide polymorphism haplotypes in the cholesteryl-ester transfer protein (CETP) gene and lipid phenotypes. Hum.Hered. 54, 166-173.

Nothnagel, M., Fürst, R. and Rohde, K. (2002). Entropy as a Measure for Linkage Disequilibrium over Multilocus Haplo- type Blocks. Hum Hered 54, 186-198.

Zdobnov, E.M., von-Mering, C., Letunic, I., Torrents, D., Suyama, M., Copley, R.R., Christophides, G.K., Thomasova, D., Holt, R.A., Subramanian, G.M., Mueller, H.M., Dimo- poulos, G., Law, J.H., Wells, M.A., Birney, E., Charlab, R., Halpern, A.L., Kokoza, E., Kraft, C.L., Lai, Z., Lewis, S., Louis, C., Barillas-Mury, C., Nusskern, D., Rubin, G.M., Salzberg, S.L., Sutton, G.G., Topalis, P., Wides, R., Wincker, P., Yandell, M., Collins, F.H., Ribeiro, J., Gelbart, W.M., Kafatos, F.C. and Bork, P. (2002). Comparative genome and proteome analysis of Anopheles gambiae and Drosophila melanogaster. Science 298, 149-159.

Structure of the Group

Group Leader Guest Scientists Prof. Dr. Jens G. Reich Dr. Ionela Oancea Dr. Mauro Santibanez-Koref* Scientists Dr. Miguel Andrade* Graduate and Undergraduate Students Dr. Peer Bork Anja Bauerfeind Ralf Bortfeldt* Katrin Hafez* Dr. Francesca Ciccarelli Jan Korbel* Dr. Stefan Dahm* Steffen Schmidt* Dr. Tobias Doerks* Parantu Shah* Robert Fürst Inga Zastrow* Dr. Wilfried Gunia Alexander Herrmann Technical Assistants Dr. Lars Juhl Jensen* Tatjana Luganskaja Andreas Kuntzagk* Anita Nothnagel* Dr. Warren Lathe* Gudrun Nürnberg Dr. Hans Lucius Brunhilde Poppe Dr. Christian von Mering Verena Thiele Dr. Ferdinand Moldenhauer* Edelgard Wolf Michael Nothnagel* Harald Pankow* Secretariat Dr. Carolina Perez-Iracheta* Veronika Heinze* Birgit Pils* Dr. Heike Pospisil * part of the period reported Dr. Florian Raible* Dr. Klaus Rohde Dr. Jürgen Rose* Dr. Franz Rüschendorf Dr. Willy Schmidt Daniel Schober Dr. Stefan Schuster* Bernd Simon* Dr. Mikita Suyama* Dr. Holger Thiele* Alexej Tschapek Dr. Athanasios Vergopoulos* Dr. Evgeny Zdobnov* 56

Gene Mapping and Identification subsequently required to identify genetic loci that contribute in Monogenic and Multifactorial to a complex disease. For this purpose, we have established Diseases the necessary techniques and emphasised automation of the experimental procedures. Our annual capacity is currently about 4,000,000 high-quality genotypes and is planned for Peter Nürnberg expansion. Mapping is now mainly based upon genotyping of SNPs. Additionally, the laboratory is equipped for the large- scale analysis of highly informative microsatellite markers. Six scientists are involved in project management, genotyping, and technology development. Two scientists concen- trate on laboratory information management (LIM) which involves the integration of genotype and phenotype data and the processing of these data for biostatistical analyses. This is done in close collaboration with the bioinformatics group (Dr. K. Rohde) and the University of Bonn (Prof. T. Wienker) who also manage the data analysis.

In a European collaborative study on the genetics of atopic dermatitis, a large number of families with two or more affected siblings were collected. A major susceptibility locus identified on chromosome 3q21 as well as further candidate regions are being investigated in these subjects. In a second Positional cloning is now widely used for the identification of European collaborative study, families are being investigated gene defects that underlie inherited diseases. A necessary first to identify genetic factors for susceptibility to common idio- step for positional cloning is the mapping of the gene locus pathic generalised epilepsies (IGE). A novel IGE susceptibi- that co-segregates within families with a particular disease or lity locus on chromosome 3q26 and suggestive loci on trait, which allows the allocation of a specific chromosomal chromosomes 14q23 and 2q36.1 are currently being pursued position to the responsible gene. Although mapping was ini- further with refined mapping and testing of positional candi- tially developed for monogenic traits, it has now become the date genes. Thus, we expect to gain important insights into most widely used strategy to locate genetic factors involved the aetiology of both disease groups. in the aetiology of multifactorial diseases. The most powerful technique currently available is linkage analysis with highly The identification of risk factors for complex traits is often polymorphic microsatellite markers, which involves an facilitated by the analysis of pedigrees from isolated popu- examination of the entire genome with a set of evenly spaced lations and takes advantage of the restricted genetic hetero- markers. This type of study is usually referred to as whole geneity in these populations. Ongoing studies include geno- genome scan. typing of a study on the genetic factors in hypertension in collaboration with the Franz-Volhard Clinic in Berlin-Buch The Gene Mapping Centre is a specialised laboratory for such (Knoblauch et al. in press). Moreover, a locus for essential high-throughput genotyping for gene mapping of monogenic (primary) hypertension was mapped to chromosome 12p in a as well as multifactorial diseases. We have developed various Chinese pedigree in collaboration with the MDC research sets of well-established markers including both microsatelli- group for Experimental Genetics of Cardiovascular Diseases tes and single-nucleotide polymorphisms (SNPs). Different (Gong et al. 2003). This locus appears to be of relevance for marker densities are used to accommodate the requirements the identification of mechanisms leading to primary hyperten- of special study designs. The laboratory is mainly funded sion and of factors for cardiovascular morbidity and morta- through grants from the German Federal Ministry of Educa- lity. Running costs for all the studies are funded through tion and Research (BMBF). Since 2001, we have participated additional external grants. in the National Genome Research Network (NGFN) as one of the major core facilities. Additional funding is provided from research grants of the DFG and through a strategy fund Mapping of monogenic diseases project (Genetics of Complex Diseases) from the Helmholtz Society of National Research Centres. The laboratory is open In contrast to multifactorial diseases, mapping of monogenic for mapping projects of other groups from Germany and traits requires less genotyping effort. Usually, it is sufficient abroad. to analyse 30 probands or less. The statistical evaluation is different and often requires skilled interpretation, for instance haplotyping. In the eight years of the existence of the lab, Mapping of multifactorial diseases more than 60 monogenic traits have been mapped in humans. For several of these the underlying gene defect has been The main focus of the Gene Mapping Centre is mapping of identified, completing the process of positional cloning. The genetic factors in multifactorial diseases. This type of study identification of mutations in ANKH, the human ortholog of involves the analysis of large numbers of phenotypically well the mouse progressive ankylosis gene, in patients with cranio- characterised families. Hundreds of markers are used for metaphyseal dysplasia (CMD) led to the investigation of so- genotyping and sophisticated biostatistical analyses are called ‘idiopathic’ infantile arterial calcification (see figure). 57

Manifestations of IIAC. a, Echocardiogram (parasternal view) of the affected girl from family 3 at age 5 days showing bright echogenic walls of the aortic arch and descending aorta (arrows), consistent with aortic calcifications. b, Native coronal CT scan of the thorax, abdomen and pelvis of the affected boy from family 4 at age 11 days. Note calcification of the entire aorta (arrow) and iliac arteries bilaterally. c, Radiogram of the right arm of the affected girl from family 5 at age 10 days. Note extensive peri-articular calcification of the elbow (arrowhead) and calcified brachial artery (arrows). d, Section through the left ventricle of the heart of patient 6, who died at age 45 days. Note hemorrhagic ischemic necrosis at the apex of the left ventricle and pallor of the subendocardial wall representing bland coagulative ischemic necrosis. A cross section of the aorta laid adjacent to the apex shows striking mural thickening and luminal narrowing. e, Cross section through the epicardial left anterior descending coronary artery from the same patient showing extensive calcifications of the internal elastic lamina (arrows) and massive myointimal proliferation (haematoxylin -eosin, 20X). f, Electron micrograph of the mineralized internal elastic lamina of the carotid artery from the same patient showing mineral deposition (arrows) with extracellular collagen fibrils and elastic fibers.

The genetic basis for the disease was disclosed by identifying orders of Keratinisation). Mal de Meleda, an autosomal reces- underlying mutations in ENPP1, the gene for ectonucleotide sive palmoplantar keratoderma, was named after an island on pyrophosphatase/phosphodiesterase 1 (Rutsch et al. 2003). the coast of Croatia where the high frequency of the disease is The gene product is a transmembrane glycoprotein involved based on a founder effect. However, mutations in the underly-

in the metabolism of inorganic pyrophosphate (PPi). The ing gene, which is related to cytotoxins of snakes, show clear work on the further characterisation of CMD and related dis- allelic heterogeneity (Eckl et al. 2003). The molecular charac- orders is funded within the SFB 577 (Molecular Basis of terisation of such rare keratinisation disorders gives insight Clinical Variability in Mendelian Disorders). Understanding into processes of epidermal differentiation and provides this group of monogenic diseases will provide us with novel models for more frequent, complex genetic skin diseases. insights into more general processes controlling bone density and may offer new approaches to the therapy of osteoporosis. Several pedigrees are being investigated with various here- ditable types of cardiomyopathy including dilated and hyper- Another focus of the group is the molecular characterisation trophic forms. Because of the heterogeneity of these dis- of hereditary skin diseases. Autosomal recessive congenital orders, the study is based on linkage analysis in single ichthyosis, a severe genodermatosis characterised by scaling pedigrees and candidate gene analysis. In the gene CRP3 that of the skin on the complete body surface, is both clinically encodes muscle LIM protein, mutations were found in and genetically heterogeneous. The identification of further patients with familial hypertrophic cardiomyopathy but not loci and the molecular characterisation of different pathways with dilated cardiomyopathy (Geier et al. 2003). CRP3 was leading to the disease is a goal of a new BMBF-funded net- chosen as a candidate gene because muscle LIM protein defi- work (Network Rare Diseases: Ichthyoses and Related Dis- cient mice exhibited a disruption of cardiac cytoarchitectural 58

organisation and developed a marked cardiac hypertrophy Dr. Hans Christian Hennies reaction and dilated cardiomyopathy. Dealing with pheno- Dr. Birgit Meyer types comprising extensive heterogeneity is also relevant to Dr. Thomas Sander* various diseases associated with renal anomalies. Several loci Dr. Eun-Kyung Suk* for nephronophthisis and related nephritic disorders were Dr. Holger Thiele* mapped in the Centre (Ruf et al. 2003). Dr. Mohammad Reza Toliat Dr. Kathrin Saar* In addition, we have continued mapping monogenic traits in animal models, mainly mice and rats. Several spontaneous Guest Scientists and ENU induced mutants were mapped, and in many cases, Dr. Regina Betz* the underlying mutations were identified. The majority of the Sebahattin Cirak* projects from external laboratories originated from Germany Dr. Sabine Haßfeld* but also from England, France, The Netherlands, Canada, the Dr. Marco Henneke* U.S., the Republic of South Africa, the United Arab Emirates, Dr. Katrin Hoffmann Australia, and other countries. Zhenyu Ju* Prof. Dr. Young-Ae Lee Dr. Ingo Marenholz* Selected Publications Dr. Dalila Pinto* Dr. Rainer Ruf* Eckl KM, Stevens HP, Lestringant GG, Westenberger- Dr. Ulrike Tauer* Treumann M, Traupe H, Hinz B, Frossard PM, Stadler R, Dr. Cilla Söderhall* Leigh IM, Nürnberg P, Reis A, Hennies HC (2003) Mal de Dr. Birgit Uhlenberg* Meleda (MDM) caused by mutations in the gene for SLURP- Silke Weixler* 1 in patients from Germany, Turkey, Palestine, and the United Dr. Matthias Wolf* Arab Emirates. Hum Genet 112, 50-56. Doctoral Students Technical Assistants Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Anja Brinckmann Francoise André Hoffmann K, Pilz B, Martiniak Y, Gehmlich K, van der Ven Katja-Martina Eckl* Christian Becker PF, Fürst DO, Vornwald A, von Hodenberg E, Nürnberg P, Patricia Entz* Ingelore Bäßmann Scheffold T, Dietz R, Osterziel KJ (2003) Mutations in the Mandy Lehmann* Carolin Engel* human muscle LIM protein gene in families with hyper- Kirsten Lenzen* Elisabeth Kirst trophic cardiomyopathy. Circulation 107, 1390-1395. Inés Mácha* Susanne Lorenz* Wenke Seifert* Barbara Lucke* Gong M, Zhang H, Schulz H, Lee YA, Sun K, Bähring S, Luft Manuela Wirsching* Marc Oliver Nätebus* FC, Nürnberg P, Reis A, Rohde K, Ganten D, Hui R, Hübner Regina Pospiech N (2003) Genome-wide linkage reveals a locus for human Graduate and Susanne Schmidt* essential (primary) hypertension on chromosome 12p. Hum Undergraduate Students Madeleine Skorna Mol Genet 12, 1273-1277. Rami Aboujamra* Inka Szangolies Carmen Bednorz* Jana Thiede* Ruf RG, Berkman J, Wolf MT, Nürnberg P, Gattas M, Ruf Jan Clemens* Nadine Wittstruck* EM, Hyland V, Kromberg J, Glass I, Macmillan J, Otto E, Ilka Diester* Nürnberg G, Lucke B, Hennies HC, Hildebrandt F (2003) A Christian Eckl* Guest Assistants gene locus for branchio-otic syndrome maps to chromosome Björn Fischer* Sabine Enigk* 14q21.3-q24.3. J Med Genet 40, 515-519. Malenka Gedicke* Heike Fischer Mandy Grusser Jenny Pech Rutsch F, Ruf N, Vaingankar S, Toliat MR, Suk A, Höhne W, Lisa Haucke* Heide Ritter* Schauer G, Lehmann M, Roscioli T, Schnabel D, Epplen JT, Daniel Herbst* Elena Schmidt* Knisely A, Superti-Furga A, McGill J, Filippone M, Sinaiko Tino Köhler* Sabrina Schulz* AR, Vallance H, Hinrichs B, Smith W, Ferre M, Terkeltaub R, Julia Krause* Monika Schwarz Nürnberg P (2003) Mutations in ENPP1 are associated with Katarina Lehmann* ‘idiopathic’ infantile arterial calcification. Nat Genet 34, 379- Nico Martin* Secretariat 381. Sandra Muschiol* Kornelia Dokup Dietlind Pachale* Nico Ruf* * part of the period reported Structure of the Group Christian Schumi* Andreas Quandt* Group Leader Denise Zornik* PD Dr. Peter Nürnberg

Scientists Dr. Janine Altmüller* Dr. Arif Bülent Ekici* Cancer Research

Signalling Pathways, Cell Biology, and Cancer Coordinator: Walter Birchmeier

Structural and Functional Genomics Coordinator: Udo Heinemann

Tumor Immunology Coordinator: Martin Lipp 60

Cancer Research Program Krebsforschungsprogramm

Walter Birchmeier Walter Birchmeier Achim Leutz Achim Leutz Udo Heinemann Udo Heinemann Martin Lipp Martin Lipp

Cancer is a collective term for heterogeneous diseases that Krebs ist ein Sammelbegriff für heterogene Erkrankungen arise in different organs by mutations of the genome. The malignen Zellwachstums, die durch Mutationen des Genoms unifying premise of cancer cells is that they escape the natur- entstehen können. Die Vorgeschichte von Krebszellen ist al “neighborhood watch” of growth control: cancer cells ähnlich: Krebszellen entziehen sich der natürlichen „Überwa- divide in an uncontrolled fashion, they escape natural cell chung“ durch Nachbarzellen. Sie teilen sich unkontrolliert, death, and the control of the immune system, and settle and werden unsterblich, schirmen sich von der Kontrolle des grow elsewhere in the body by migrating through the blood Immunsystems ab, wandern über das Blut- und Lymphsystem and lymph system (metastasis). The objective of the MDC und siedeln an verschiedenen Körperstellen, um Tochter- Cancer Research Program is to understand how cancer deve- geschwulste (Metastasen) zu bilden. Das Ziel des MDC- lops and progresses and to use that knowledge to improve the Krebsforschungsprogramms besteht darin zu verstehen, wie diagnosis and, ultimately, the treatment of cancer. Krebs sich entwickelt und fortschreitet, um diese Kenntnisse zur Verbesserung der Diagnose und auch zur Behandlung von How does cancer arise? The human genome consists of be- Krebs anwenden zu können. tween 30,000 to 40,000 genes. Some of these genes are of particular importance for the regulation of cellular behavior. Many Wie entsteht Krebs? Das menschliche Genom besteht aus of these crucial genes are recurring targets of genetic altera- 30.000 bis 40.000 Genen. Einige dieser Gene sind für die tions that provoke the emergence of cancer. These genes are Regulation des Zellverhaltens von besonderer Bedeutung. categorized as either “oncogenes” or “tumor suppressor genes” Viele dieser Schlüsselgene sind wiederkehrende Ziele gene- and code for regulatory and structural proteins that control cell tischer Veränderungen, die die Entstehung von Krebs hervor- proliferation, differentiation, apoptosis, and cell migration. rufen. Diese Gene werden als „Onkogene“ oder „Tumor-Sup- Many of them are also active during embryogenesis or during pressor“-Gene kategorisiert und kodieren Proteine, die die the development of distinct cell types. This is why tumor cells Zellteilung, -differenzierung, -apoptose und -wanderung kon- appear to share characteristics of embryonic cells. trollieren. Viele dieser Gene sind auch während der Embryo- genese oder der Entwicklung bestimmter Zelltypen aktiv. The MDC Cancer Research Program consists of several scientific research groups that work in the fields of signal Das Krebsforschungsprogramm des MDC setzt sich aus meh- transduction and growth control, structural genome research, reren wissenschaftlichen Gruppen zusammen, die auf den and of tumor immunology. Knowledge in various basic bio- Gebieten der Signalumwandlung und Wachstumskontrolle, medical and clinical disciplines is pooled to investigate the der Strukturgenom-Forschung und der Tumorimmunologie causes and the emergence of cancer and to find rational treat- tätig sind. Diese Untersuchungen werden in enger Zusam- ments. Cancer studies are conducted in close collaboration menarbeit mit den klinisch orientierten Gruppen der Robert- with clinically orientated groups at the Robert-Roessle Roessle-Krebsklinik der Charité/Humboldt-Universität Berlin Cancer Clinic of the Charité/Humboldt University in Berlin, und der Helios-Klinik durchgeführt. Das Ziel des Krebs- and at the Helios Clinic. The aim of the Cancer Research forschungsprogramms des MDC besteht darin, Gene zu ent- Program is to discover and to characterize genes that are re- decken und zu charakterisieren, die für das Entstehen von sponsible for the emergence of cancer and to determine how Krebs verantwortlich sind und zu ermitteln, welche Rolle these gene products function in the above-mentioned crucial diese Genprodukte in den o. g. entscheidenden Zellprozessen cellular processes and during progression of the disease. The sowie im weiteren Verlauf der Krebserkrankung spielen. Das resulting knowledge lays the essential groundwork for the hieraus resultierende Wissen bildet den Grundstein für die development of future cancer treatments. Entwicklung zukünftiger Krebsbehandlungen. 61

Signaling Pathways, Cell Biology, and Cancer Signalwege, Zellbiologie und Krebs

Epithelial morphogenesis and differentiation have been ana- Morphogenese und Differenzierung von Epithelzellen wur- lyzed by defining the adhesive and signaling capacities of the den anhand der Haft- und Signaleigenschaften des E-Cad- E-cadherin/catenin/Wnt system (W. Birchmeier). In addition, herin/Catenin/Wnt-Systems analysiert (W. Birchmeier). the role of the scatter factor/hepatocyte growth factor and its Außerdem wurde die Rolle des Scatterfaktors/Hepatozyten- receptor, the Met tyrosine kinase, has been examined in the Wachstumsfaktors und seines Rezeptors, der Met-Tyrosin- morphogenesis of epithelial cells. Several components of the kinase, in der Morphogenese von Epithelzellen analysiert. In Wnt- and Met pathways have been found to be mutated in a einer Vielzahl menschlicher Tumore wurden Mutationen variety of human tumors. Several new members of these mehrerer Komponenten der Wnt- und Met-Signalwege fest- pathways have been discovered in this laboratory (for exam- gestellt. Mehrere neue Komponenten dieser Signalwege (wie ple, Lef-1, Conductin, Diversin, Gab1, Dok’s, and Hakai). z. B. Lef-1, Conductin, Diversin, Gab1, Dok’s und Hakai) The function of these novel proteins is currently investigated sind in diesem Labor ermittelt worden. Die Funktionsweise by genetic means in mice and in human tumors. Mouse dieser neu entdeckten Proteine wird derzeit mit genetischen models are generated which reflect particular genetic alter- Mitteln in Mäusen und in menschlichen Tumoren untersucht. ations found in human cancers. It was recently found that Mausmodelle werden hergestellt, die besondere, in mensch- -catenin, a component of the Wnt pathway, is a key regula- lichen Tumoren vorgefundene genetische Veränderungen tor of the formation of the apical ectodermal ridge (AER) and widerspiegeln. So wurde kürzlich durch die konditionelle of the dorsal-ventral axis of vertebrate limbs by conditional Inaktivierung von Genen in Mäusen festgestellt, dass -Ca- gene deletion in mice. It was demonstrated that -catenin acts tenin, eine Komponente des Wnt-Signalweges, ein Schlüssel- downstream of the BMP receptor IA in AER induction, but regulator für die Bildung der embryonalen apikal-ektoderma- upstream or parallel to this receptor in dorsal-ventral pattern- len Leiste (AER) und der dorsal-ventralen Achse der ing by generation of compound mutants (Soshnikova et al., Gliedmaßen ist. Es konnte belegt werden, dass -Catenin in Genes & Development 17, 1963-1968, 2003). der AER-Induktion unterhalb des BMP-Rezeptors IA, in der dorsal-ventralen Musterung aber oberhalb dieses Rezeptors The reason why cancers of similar stage and histomorpho- aktiv ist (Soshnikova et al., Genes & Development 17, 1963- logical characteristics exhibit variable clinical outcomes 1968, 2003). remains unknown. The genetic reasons underlying differences in the aggressiveness of local tumors growth, in the Die Ursache dafür, dass verschiedene Krebsformen ähnlicher ability of tumors to metastasize or in the susceptibility to Stadien und histomorphologischer Eigenschaften variable chemo- and radiotherapy also remain largely unknown. klinische Resultate zeigen, ist nicht geklärt. Auch die gene- Microarray and differential display techniques have been tischen Gründe, die für Unterschiede in der Aggressivität des used (P. Schlag/W. Birchmeier) to identify genes that are re- lokalen Tumorwachstums, der Fähigkeit von Tumoren zur sponsible for variable tumor behavior (of primarily colorectal Metastasenbildung oder ihrer Empfänglichkeit gegenüber cancers, breast cancers, and sarcomas). The Robert-Roessle Chemo- und Radiotherapie ausschlaggebend sind, sind wei- Clinic at the MDC has a large tissue and serum bank, consist- testgehend unbekannt. Um Gene zu identifizieren, die für va- ing of more than 10,000 tissue samples derived from human riables Tumorverhalten verantwortlich sind, wurden Mikro- primary tumors, their metastases, and from corresponding array- und „Differenzialdisplay“-Verfahren bei kolokteralen normal tissues. This bank forms the backbone of the described Tumoren, Brustkrebs und Sarkomen angewandt (P. Schlag/ research programme. W. Birchmeier). Die Robert-Roessle-Klinik am MDC besitzt eine große Gewebe- und Serumbank mit über 10.000 Gewe- The emergence of leukemia is connected to the deregulation beproben, die von menschlichen Primärtumoren, ihren Meta- and mutation of genes that encode critical regulators involved stasen und von entsprechenden Normalgeweben stammen. in hematopoietic stem cell biology and in blood cell differentiation. Transcription factors of the C/EBP family, c-Myb, Das Auftreten von Leukämie steht in Verbindung mit der and Scl/Tal1 balance the finely tuned system of stem cell self- Deregulation und Mutation von Genen, deren Produkte die renewal (A. Leutz). Mutations in these genes may cause leu- Blutzelbildung und Blutzelldifferenzierung bestimmen. Die kemic conversion in these cells. It was found recently that the Transkriptionsfaktoren der C/EBP-Familie sowie c-Myb und mRNAs for C/EBP, C/EBP, and Scl/Tal1 give rise to Scl/Tal1 kontrollieren die fein abgestimmte Balance zwi- alternatively initiated protein isoforms with largely different schen Stammzellen und Zelldifferenzierung (A. Leutz). Mu- functions in growth control and cell differentiation. Alterna- tationen in diesen Genen können zu Leukämien führen. Es tively initiated Scl/Tal1 proteins may induce differentiation wurde kürzlich festgestellt, dass die Expression der C/EBP-, towards various blood cell lineages (Calkhoven et al., Genes C/EBP- und Scl/Tal1-Proteine durch differentielle Initiation & Development, 17, 959-964, 2003). Furthermore, the func- ihrer mRNA-Translation reguliert sind. Alternativ initiierte tion of C/EBP is regulated by the oncogenic ras signaling Scl/Tal1-Proteine können die Differenzierung in verschie- pathway. Ras signals determine whether C/EBP acts as an dene Blutzelllinien hervorrufen (Calkhoven et al., Genes & activator or as a repressor of genes by regulating the interac- Development, 17, 959-964, 2003). Zudem wurde erkannt, tion of C/EBP with active and/or repressive “Mediator” dass die Funktion des C/EBP, welches in vielen Tumoren complexes. Mediators are large multi-protein complexes exprimiert ist, durch das ras-Tumorprotein reguliert wird. found in all eukaryotes that link transcription factors to the Ras-Signale bestimmen, ob C/EBP als Aktivator oder basic transcription machinery (Mo et al., Molecular Cell, 13, Repressor von Genen agiert, indem sie die Interaktion des 1-10, 2004). C/EBP mit aktiven oder mit repressiven „Mediator“-Kom- 62

An example of signal-dependent gene regulation with an plexen regulieren. Mediator-Komplexe sind große Multi- extensive medical relevance is the nuclear factor B (NF-B) proteinkomplexe, die in allen Eukaryoten vorkommen und transcription factor family (C. Scheidereit). NF-B is essential Transkriptionsfaktoren mit der basalen Transkriptions- for innate and adaptive immunity, but also plays important maschinerie verknüpfen (Mo et al., Molecular Cell, 13, 1-10, roles in cell proliferation, programmed cell death, and in 2004). early embryogenesis. NF-B has been found to contribute to the development of diseases such as cancer, various Die Familie des Kernfaktors NF-B ist ein Beispiel für die inflammatory disorders, and skin diseases. NF-B activation signalabhängige Genregulierung einer Transkriptionsfaktor- pathways are analyzed biochemically and in mouse models. Familie, die von weitreichender medizinischer Bedeutung Current emphasis has been given to the genome-wide identi- ist. NF-B spielt eine bedeutende Rolle für die angeborene fication of NF-B target genes and to the identification of und adaptive Immunität, hat jedoch auch erheblichen Ein- cross-talk with other transcription factors and signaling fluss auf die Zellproliferation, den programmierten Zelltod pathways (Hinz, M., et al., J. Exp. Med. 196, 605-17, 2002). und die frühe Embryogenese (C. Scheidereit). NF-B trägt Mouse models for human disease have been generated. A nachweislich zur Entwicklung von Krankheiten wie der crosstalk between the NF-B pathway and JUN proteins in Tumorgenese, verschiedenen Entzündungsleiden und Haut- lymphomagenesis has recently been established. c-JUN was krankheiten bei. Die NF-B-Aktivierungswege werden bio- shown to be activated by autonomous pathways, with NF-B chemisch und in Mausmodellen analysiert. Besonderer playing a role in determining the composition of the AP-1 Nachdruck wurde in letzter Zeit auf die genomweite Identi- subunit by upregulating JunB. AP-1 cooperates with NF-B fizierung von NF-B-Zielgenen und den Nachweis des at target gene promoters. c-Jun and JunB overexpression is Zusammenspiels mit anderen Transkriptionsfaktoren und observed in the great majority of classical Hodgkin’s tumors, Signalwegen gelegt (Hinz, M., et al., J. Exp. Med. 196, 605- and to a lesser extent in anaplastic large cell lymphomas but 17, 2002). Ein Zusammenwirken zwischen NF-B- und den not in other B- or T-cell malignancies (Krappmann et al., JUN-Proteinen in der Lymphomagenese wurde kürzlich EMBO J. 21, 4104-4113, 2002). nachgewiesen. Dabei ergab es sich, dass c-JUN von autono- men Signalwegen aktiviert wird, wobei NF-B eine Rolle Rapid and specific proteolysis via the ubiquitin-proteasome bei der Bestimmung der Zusammensetzung der AP-1-Unter- pathway is a key step in many regulatory processes and also einheit spielt, indem es JunB hochreguliert. AP-1 kooperiert important for the elimination of mis-folded proteins. Defects zudem mit NF-B an Zielgen-Promotern. Eine Überexpres- in this pathway are associated with the development of sion von c-Jun und JunB wird in der überwiegenden Mehr- disease including cancer and viral infections (T. Sommer). heit klassischer Hodgkin-Tumore und in geringerem Maße Components and underlying principles of the evolutionary in anaplastischen, großzelligen Lymphomen, jedoch nicht in highly-conserved ubiquitin system are being studied. Re- anderen B- oder T-Zelltumoren beobachtet (Krappmann et search focuses mainly on the compartmentalized functions of al., EMBO J. 21, 4104-4113, 2002). the system, which bring together the fields of specific proteolysis and of intracellular protein transport and secretion. Die schnelle und spezifische Proteolyse über das Ubiquitin- Endoplasmic reticulum (ER) associated protein degradation Proteasom ist ein Schlüsselschritt für viele Regulationspro- by the ubiquitin proteasome system requires dislocation of zesse und von zentraler Bedeutung für die Eliminierung fehl- the proteolytic substrates from the ER into the cytosol. gefalteter Proteine. Defekte in diesem Signalweg sind für die Recent analyses revealed that the AAA ATPases of the 26S Entwicklung von Krankheiten einschließlich Krebs und proteasome are not directly involved in exporting an ER- Virusinfektionen verantwortlich (T. Sommer). Bestandteile luminal substrate. Instead, a related AAA ATPase-complex, und grundlegende Prinzipien des evolutionär hochkonser- Cdc48p/p97, plays a crucial role in ER associated protein vierten Ubiquitin-Systems werden derzeit untersucht. Die degradation upstream of the proteasome (Jarosch, E., Nature Forschung konzentriert sich hauptsächlich auf die komparti- Cell Biol. 4, 134-139, 2002). mentalisierten Funktionen des Systems, die die Bereiche der spezifischen Proteolyse, des intrazellulären Proteintransports Tight control over initiation of DNA replication is of central und der Sekretion verknüpfen. Die durch das Ubiquitin- importance for cell proliferation to ensure normal develop- Proteasomsystem hervorgerufene Proteindegradation steht ment and differentiation. It is still not fully understood how mit dem endoplasmatischen Retikulum (ER) in einem engen particular DNA sequences regulate this process, whereas the Zusammenspiel und erfordert eine Dislokation der proteolyti- function of proteins involved in this process in higher eukary- schen Substrate aus dem ER in das Zytosol. Neuerliche Ana- otes have begun to emerge. Aspects of replication initiation in lysen haben ergeben, dass die AAA ATPasen des 26S Protea- both Drosophila and in mammalian cells are being studied soms nicht direkt am Export eines ER-Lumensubstrats (M. Gossen). beteiligt sind. Stattdessen spielt ein verwandter AAAATPase- Komplex, Cdc48p/p97, der oberhalb des Proteasoms angreift The Sleeping Beauty transposon shows high transpositional eine entscheidende Rolle in der ER-assoziierten Proteindeg- activity in cells of vertebrate species (Z. Ivics). Thus, this radation (Jarosch, E., Nature Cell Biol. 4, 134-139, 2002). transposable element is a useful tool for genetic engineering and gene discovery in vertebrates, as well as for the dissection Für die normale Entwicklung von Zellen und für die Regula- of molecular mechanisms of transposon-host cell interactions. tion der Zellproliferation und Differenzierung ist eine strenge Depending on the insertion site, inducible transcription from Kontrolle der Initiierung der DNA-Replikation von zentraler promoters inside the transposon will overexpress and/or Bedeutung. Es ist bis heute nicht vollständig bekannt, wie be- ectopically express endogenous genes or parts of genes. This stimmte DNA-Sequenzen diesen Prozess regulieren, während 63

can be exploited for the discovery of novel oncogenes and die Funktion der Proteine, die bei höheren Eukaryoten an die- tumor suppressors using dominant screens both in tissue sem Prozess beteiligt sind, allmählich klarer wird. Aspekte culture and in living organisms. As transposition inherently der Replikationsinitiierung werden derzeit sowohl an Droso- involves the generation of DNA damage, the Sleeping Beauty phila als auch an Säugerzellen untersucht (M. Gossen). element can serve as an experimental system to study the basic molecular mechanisms involved in DNA repair. It has recently Ein besonderes genetisches Element (genannt Sleeping been shown that non-homologous end-joining and homolo- Beauty-Transposon) weist eine hohe transpositionelle Akti- gous recombinational repair pathways both contribute to the vität in Zellen von Wirbeltieren auf (Z. Ivics). Daher ist repair of transposition-induced DNA damage. Thus, important dieses Transposon Element ein hilfreiches Werkzeug der similarities as well as differences exist between cellular re- Gentechnik zur Entdeckung von Genen in Wirbeltieren, sponses to V(D)J recombination, retroviral integration, and sowie zur Analyse molekularer Mechanismen von Transpo- DNA transposition (Izsvak et al., Mol. Cell 9, 147-156, 2004). son-Wirt-Zellinteraktionen. Je nach Insertionsort werden in- duzierbare Transkriptionen von Promotoren innerhalb des Transposons eine Überexpression und/oder ektopische Structural and Functional Genomics Expression endogener Gene oder Genteile bewirken. Dies kann zur Entdeckung neuer Onkogene und Tumorsuppresso- The systematic analysis of three-dimensional protein struc- ren sowohl in Gewebekulturen als auch in lebenden Organis- tures, commonly known as structural genomics, has been made men genutzt werden. Da die Transposition die Generierung possible by the completion of the human genome project and von DNA-Schäden mit sich bringt, kann das Sleeping through technical developments in recombinant protein pro- Beauty-Transposon als experimentelles System zur Erfor- duction (U. Heinemann). A consortium of research institu- schung der grundlegenden molekularen Mechanismen tions based in the Berlin-Brandenburg area has established dienen, die an der DNA-Reparatur beteiligt sind. Es konnte facilities and a technical approach that allow the structure kürzlich nachgewiesen werden, dass sowohl nicht-homologe determination of human proteins by NMR spectroscopy and Endverbindungs- als auch homologe Rekombinations-Repa- X-ray crystallography at a vastly accelerated rate and with raturwege an der Reparatur durch Transposition-induzierter greater accuracy (Heinemann et al., Acc. Chem. Res. 36, 157- DNA-Schäden beteiligt sind (Izsvak et al., Mol. Cell 9, 163, 2003). The human protein structures solved so far 147-156, 2004). include those of the translational inhibitor p14.5 (a protein that is downregulated in liver and kidney tumors), gankyrin (the product of a gene linked to hepatocellular carcinoma that Strukturelle und funktionale Genomik has been described to physically interact with the retinoblastoma protein, Rb), the cyclin-dependent protein kinases Die systematische Analyse der dreidimensionalen Strukturen 4 and 6, the 26S proteasome, and Bet3p, a central subunit of von Proteinen, allgemein bekannt als strukturelle Genomik, the vesicle-tethering TRAPP complex located at the cis-Golgi wurde durch technische Neuentwicklungen in der Produktion membrane. rekombinanter Proteine ermöglicht (U. Heinemann). Ein Konsortium von Forschungsinstitutionen aus dem Raum Computer simulations of macromolecular structures provide Berlin-Brandenburg hat entsprechende Einrichtungen und insights into their conformational transitions and dynamics– eine technische Methodik geschaffen, die die Strukturbestim- information which cannot be provided by experimental struc- mung menschlicher Proteine mittels NMR-Spektroskopie ture determination approaches (H. Sklenar). A new Monte und Röntgen-Kristallographie in wesentlich kürzerer Zeit Carlo simulation algorithm, that permits prediction of und mit erheblich größerer Präzision ermöglicht als bisher sequence-dependent structures of DNA and the study of (Heinemann et al., Acc. Chem. Res. 36, 157-163, 2003). Zu DNA-ligand interactions, has been implemented and tested. den bislang aufgeschlüsselten menschlichen Proteinstruktu- Zinc-finger proteins were identified and annotated for the ren zählen die des Translationsinhibitors p14.5 (ein Protein, completed sequenced genomes of Drosophila melanogaster das in Leber- und Nierentumoren herunterreguliert wird), and Arabidopsis thaliana in genome-wide computer searches Gankyrin (das Produkt eines mit Leberzellenkarzinomen (S. Böhm). A novel zinc-finger-associated domain (ZAD assoziierten Gens, das mit dem Retinoblastom-Protein inter- restricted to insects was identified (Chung et al., EMBO Rep. agiert), die Zyklin-abhängigen Proteinkinase 4 und 6, das 3, 1158-1162, 2002). 26S-Proteasom und Bet3p, eine zentrale Untereinheit des membranbindenden TRAPP-Komplexes, der an der cis-Golgi- Breast or ovarian cancer usually arises sporadically but a Membran angesiedelt ist. fraction (~10%) of breast-cancer cases are heritable and are caused by mutations in the tumor-suppressor genes BRCA1 Computer-Simulation der Struktur von Makromolekülen bie- or BRCA2. Additional high- and low-penetrance genes tet Einblick in deren Konformationsübergänge und Dynamik, linked to breast cancer are currently being identified by die von Methoden der experimentellen Strukturbestimmung linkage analysis in high-risk families and in association stud- nicht geliefert werden können (H. Sklenar). Ein neuer Simu- ies involving large cohorts of patients (S. Scherneck). SASH1 lations-algorithmus (Monte-Carlo), der die Vorhersage se- on chromosome region 6q24 is one of these recently identi- quenzabhängiger DNA-Strukturen und die Untersuchung der fied candidate tumor suppressor genes (Zeller et al., Onco- Interaktionen von DNA-Liganden ermöglicht, ist etabliert gene 22, 2972-2983, 2003). Some of the somatic alterations und getestet worden. Zinkfinger-Proteine wurden in einer ge- in these genes have been correlated with clinico-pathological nomweiten Computer-Suche bei Drosophila melanogaster observations. und Arabidopsis thaliana identifiziert (S. Böhm). Eine neue 64

Tumor Immunology Zinkfinger-assoziierte Domäne (ZAD) wurde identifiziert (Chung et al., EMBO Rep. 3, 1158-1162, 2002). In Hodgkin’s lymphoma, several molecular defects have been associated with the deregulation of cell proliferation, differ- Brust- oder Ovarialkarzinome treten normalerweise spora- entiation, and apoptosis (B. Dörken). Hodgkin-Reed Stern- disch auf. Ein gewisser Anteil (~ 10 %) der Brustkrebsfälle ist berg cells show a constitutive activity of transcription factors jedoch erblich bedingt und wird durch Mutationen in den such as AP-1 and NF-B. The latter signaling pathway Tumorsuppressorgenen BRCA1 oder BRCA2 verursacht. appears to be partly responsible for the apoptosis resistance Weitere Gene von hoher und geringer Penetranz, die mit of HRS cells by the upregulation of c-FLIP proteins and the Brustkrebs in Verbindung stehen, werden gegenwärtig durch inhibition of the classical cell death pathways involving eine Kopplungsanalyse in Familien mit hohem Risiko und in CD95 and TRAIL. In addition, cell proliferation and apopto- Assoziationsstudien an großen Patientengruppen identifiziert sis of HRS cells appears to be influenced by Notch signaling (S. Scherneck). Das Gen SASH1 in der Chromosomregion as the interaction between Notch1 on tumor cells and its 6q24 ist einer dieser kürzlich identifizierten Tumorsuppres- ligand Jagged1 induces proliferation and inhibition of sorgen-Kandidaten (Zeller et al., Oncogene 22, 2972-2983, apoptosis in vitro (Jundt et al. Blood 99, 3398-3403, 2002). 2003). Remarkably, ligand-induced Notch signaling was also identified as a critical growth factor for cultured and primary multiple myeloma cells suggesting that these interaction in the Tumor Immunologie context of the bone marrow micro-environment contribute to myelomagenesis in vivo (Jundt et al., Blood, published online In Hodgkin Lymphomen wird die deregulierte Proliferation, Jan. 15, 2004). Differenzierung und Apoptose von Tumorzellen mit einer Anzahl molekularer Defekte in diesen Zellen assoziiert Little is known about the mechanisms of tumor rejection and (B. Dörken). Hodgkin-Reed-Sternberg (HRS) Zellen zeigen the intricate interaction of T cells, antigen presenting cells, beispielsweise eine konstitutive Aktivität der Transkriptions- and tumor cells during this process. Inflammatory cytokines, faktoren NF-B und AP-1. Insbesondere NF-B kommt da- such as IL-4 and IFN-, are important factors influencing bei eine zentrale Funktion bei der Apoptose-Resistenz der tumor immunity (T. Blankenstein). For example, IFN- HRS-Zellen zu, die mit einer verstärkten Expression von mediated angiostasis has been shown to be a general mecha- FLIP Proteinen und der Inhibition klassischer Signalwege der nism and critical requirement for tumor rejection by CD8+ T Apoptose über CD95 und TRAIL einhergeht. Zusätzlich cells (Qin et al. Cancer Res. 63, 4095-4100, 2003). In addi- scheint die Signalvermittlung über Notch-Rezeptoren das tion, a novel mechanism has been discovered by which IL-4 Wachstum von HRS-Zellen zu beeinflussen, da die Inter- contributes to tumor rejection by acting on tumor associated aktion zwischen Notch1 auf der Oberfläche von Tumorzellen stromal cells (Schüler et al., J. Exp. Med. 198, 1487-1493, und seinem Liganden Jagged1 in vitro die Proliferation der 2003). Hodgkin-Zellen stimuliert und gleichzeitig die Apoptose inhibiert (Jundt et al. Blood 99, 3398-3403, 2002). Das Sig- Autoreactive T cells that have escaped the control mecha- naling über Notch ist darüber hinaus ein wichtiger Faktor für nisms of the immune system may cause severe autoimmune das Wachstum primärer und kultivierter Plasmozytom-Zellen diseases. In addition, genetic and poorly defined environ- und ist daher vermutlich auch im Mikromilieu des Knochen- mental factors also have an influence on the induction of marks an der Entstehung von Plasmozytomen beteiligt (Jundt these diseases (K. Falk/O. Rötzschke). Small molecular com- et al., Blood, published online Jan. 15, 2004). pounds containing H-bond donor groups have been shown to drastically accelerate the exchange of peptide antigens pre- Die molekularen Mechanismen der Tumorabstoßung, insbe- sented by MHC class II molecules on the surface of activated sondere die komplexe Interaktion von T-Zellen, Antigen-prä- antigen presenting cells (Falk et al., J. Biol. Chem. 277, 2709- sentierenden Zellen und Tumorzellen ist bislang wenig cha- 2715, 2002). This mechanism bears the risk of loading rakterisiert. Inflammatorische Zytokine wie z. B. IL-4 und activated antigen presenting cells with peptides derived from IFN- sind dabei für die Tumorimmunität von besonderer autoantigens and, consequently, results in the activation of Bedeutung (T. Blankenstein). Bei der IFN--vermittelten An- autoreactive T cells. giostase handelt es sich um einen generellen Mechanismus und eine wichtige Voraussetzung für die effiziente Tumorab- A critical aspect in cancer immunotherapy is the generation stoßung durch CD8+ T-Zellen (Qin et al. Cancer Res. 63, of a strong, tumor-specific immune response. Retrovirally 4095-4100, 2003). Darüber hinaus wurde ein neuer Mecha- transduced genetically modified T cells expressing T cell nismus entdeckt, über den IL-4 über die Tumor-assoziierten receptors specific for tumor-associated antigens may help to Stromazellen zur Tumorabstoßung beiträgt (Schüler et al., J. facilitate adoptive therapy (W. Uckert). As T cell-mediated Exp. Med. 198, 1487-1493, 2003). immune responses depend on the efficient presentation of antigen by dendritic cells, it might also be possible to Autoreaktive T-Zellen, die den Kontrollmechanismen des enhance the therapeutic efficacy of vaccination strategies by Immunsystems entkommen, können schwere Autoimmun- modulating the proliferation, differentiation, or function of erkrankungen hervorrufen. Zusätzlich sind aber auch gene- dendritic cells (A. Pezzutto). tische Faktoren und Umwelteinflüsse, wie kleine Moleküle, die bisher wenig charakterisiert sind, für den Ausbruch dieser Dendritic cells are derived from hematopoietic progenitors Krankheiten mit verantwortlich (K. Falk/O. Rötzschke). but the early steps in linage decision and dendritic cell matu- Kleine Moleküle mit funktionellen Gruppen, die als Wasser- 65

ration remained largely unknown (M. Zenke). By performing stoffbrücken-Donor wirken, beschleunigen den Austausch large-scale gene expression analysis, the inhibitory helix- von Peptiden, die durch MHC-Klasse II Moleküle auf der loop-helix (HLH) transcription factor Id2 has now been Oberfläche von aktivierten Antigen-präsentierenden Zellen identified as a major regulator of DC differentiation (Hacker präsentiert werden (Falk et al., J. Biol. Chem. 277, 2709- et al. Nat. Immunol. 4, 380-386, 2003). Mice lacking expres- 2715, 2002). Dieser Mechanismus birgt jedoch die Gefahr, sion of Id2 also lack several subsets of dendritic cells and, in dass aktivierte Antigen-präsentierende Zellen mit Autoanti- addition, the balanced expression of Id2 and an activating genen beladen werden und auf diesem Weg autoreaktive HLH factor, such as E2A, is probably important for the dif- T-Zellen aktivieren. ferentiation of the hematopoietic progenitor cells into either B cells or dendritic cells. Ein wichtiger Aspekt der Immuntherapie bei Krebs ist die Generierung einer effizienten Tumor-spezifischen Immun- Immune system homeostasis and adaptive immunity require antwort. T-Zellen, die mit Hilfe retroviraler Vektoren gene- that naïve lymphocytes constantly recirculate through sec- tisch so modifiziert wurden, so dass ihre T-Zell-Rezeptoren ondary lymphoid organs. Lymphocyte and dendritic cell spezifisch ein Tumor-assoziiertes Antigen erkennen, sind für entry into the T and B cell zones of secondary lymphoid eine adoptive T-Zell-Therapie besonders geeignet (W. organs is largely regulated by the chemokine receptors Uckert). Da eine T-Zell vermittelte Immunantwort von einer CXCR5 and CCR7. Moreover, it has been shown that the effektiven Präsentation des Antigens durch dendritische Zel- balance of responsiveness to chemokine ligands for CXCR5 len abhängt, kann die Effizienz von Vakzinierungsstrategien and CCR7, which are made in separate but adjacent zones, über die Modulation der Proliferation, Differenzierung oder mediates B cell relocalization in response to antigen (Reif et Funktion dendritischer Zellen verbessert werden (A. Pez- al., Nature 416, 94-99, 2002). Mice deficient for the chemo- zutto). kine receptor CXCR5 lack several lymph nodes and Peyer’s patches. Although lymphoid organ development is unaffected Über die Differenzierung dendritischer Zellen aus hämato- in CCR7-deficient mice, it turned out that both chemokine poietischen Vorläuferzellen ist noch wenig bekannt (M. receptors cooperate in lymph node development as mice Zenke). Mit Hilfe einer Microarray-basierten Analyse der double-deficient for CXCR5 and CCR7 lack all but mesen- Genexpression dendritischer Zellen wurde Id2, ein inhibitori- teric lymph nodes (Müller et al., Immunol. Rev. 195, 117- scher Helix-loop-helix (HLH) Transkriptionsfaktor, als ein 135, 2003). zentraler Faktor für die Differenzierung dendritischer Zellen identifiziert (Hacker et al. Nat. Immunol. 4, 380-386, 2003). Anscheinend ist die Balance der Expression von Id2 und eines aktivierenden HLH Proteins, wie z. B. E2A, für die Richtung der Differenzierung der Vorläuferzellen in B-Zellen oder dendritische Zellen von Bedeutung.

Die physiologische Migration von Lymphozyten sowie die Einwanderung von Lymphozyten und dendritischen Zellen in die T- und B-Zell Zonen sekundärer lymphatischer Organe wird maßgeblich durch die Chemokinrezeptoren CXCR5 und CCR7 gesteuert. Darüber hinaus sind diese Rezeptoren auch für die präzise Lokalisierung von B-Zellen in den Mikrokom- partimenten sekundärer lymphatischer Organe verantwortlich, indem die Responsivität gegenüber den Liganden von CXCR5 und CCR7 moduliert wird (Reif et al., Nature 416, 94-99, 2002). CXCR5-Knock-out-Mäusen fehlen verschiedene periphere Lymphknoten, während die Entwicklung von Lymphknoten in CCR7-Knock-out-Mäusen kaum betroffen ist. Überaschenderweise zeigte sich bei der Analyse von Dop- pel-Knock-out-Mäusen für CXCR5 und CCR7, dass beide Rezeptoren in der Entwicklung von Lymphknoten kooperie- ren, da diese Mäuse bis auf die mesenterialen keine weitern Lymphknoten ausbilden (Müller et al., Immunol. Rev. 195, 117-135, 2003). Signalling Pathways, Cell Biology, and Cancer

Epithelial Signal Transduction, Hakai, a c-Cbl-like protein, ubiquinates and Invasion, and Metastasis induces endocytosis of the E-Cadherin complex Yasuyuki Fujita and Dietmar Zechner. In collaboration with Thomas Sommer (MDC), Gerd Krause (FMP Berlin), Martin Walter Birchmeier Scheffner (Univ. Köln), and Jürgen Behrens (Univ. Erlangen).

In epithelial cells, tyrosine kinases induce tyrosine phosphorylation and ubiquitination of the E-cadherin complex, with results in endocytosis of E-cadherin. With a modified yeast 2-hybrid system, we isolated Hakai, an E-cadherin binding protein, which is an E3 ubiquitin-ligase. Hakai contains Our laboratory concentrates on the molecular analysis of SH2, RING, zinc-finger and proline-rich domains and inter- epithelial morphogenesis and differentiation. In previous acts with E-cadherin in a tyrosine phosphorylation-dependent years, we defined the adhesion and signaling capacities of the manner, inducing ubiquitination of the E-cadherin complex. E-cadherin/catenin/Wnt system. Moreover, we have investi- Expression of Hakai in epithelial cells disrupts cell-cell gated the role of scatter factor/hepatocyte growth factor contacts and enhances endocytosis of E-cadherin and cell (SF/HGF) and its receptor, the c-met tyrosine kinase, in motility. Through dynamic recycling of E-cadherin, Hakai morphogenesis of epithelial cells. Components of the Wnt can thus modulate cell adhesion and could participate in the and c-met pathways are mutated in a variety of human regulation of epithelial-mesenchymal transitions in develop- tumors. ment or metastasis.

Epithelial cells can loose expression of E-cadherin during tumor progression and this loss correlates with the appea- Genetic interaction between Wnt/-catenin and rance of highly invasive carcinoma cells. We have recently BMP receptor signaling during formation of the identified the new E3 ubiquitin ligase, Hakai, which results in AER and the dorsal-ventral axis in the limb E-cadehrin degradation in a tyrosine-phosphorylation depen- Natalia Soshnikova, Dietmar Zechner and Jörg Hülsken. In dent manner (Fujita et al., 2002). The function of cadherins collaboration with Richard Behringer (Univ. Texas, Houston), depends strictly on cytoplasmic linkage molecules, -catenin, Makoto Taketo (Univ. Kyoto), and Brian Crenshaw (Univ. Phi- plakoglobin, p120, which mediate interaction of cadherins ladelphia). with the cytoskeleton. We have also shown that -catenin binds to the transcription factor LEF-1/TCF and that this in- By conditional gene ablation in mice, we found that -catenin teraction translocates -catenin to the cell nucleus and regu- is a key regulator of the formation of the apical ectodermal lates gene expression (Behrens et al., 1996). This provides a ridge (AER) and of the dorsal-ventral axis of the limbs. By molecular mechanism for the transmission of Wnt signals to generation of compound mutants, we also show that -cate- the cell nucleus, which is essential in many developmental nin acts downstream of the BMP receptor IA in AER induct- processes and in tumor progression (Huelsken et al., 2001; ion but upstream or parallel in dorsal-ventral pattering (see Soshnikova et al., 2003; Morkel et al., 2003). In the absence Figure). Thus, AER formation and dorsal-ventral pattering of of Wnt signals, -Catenin is degraded by the Axin/Conduc- limbs are tightly controlled by an intricate interplay between tin/GSK3/CK1 system (Behrens et al., 1998; Schwarz- Wnt/-catenin and BMP receptor signaling. Romond et al., 2002).

The scatter factor/c-met system transduces various signals in The ankyrin repeat protein Diversin recruits Casein epithelial cells, such as scattering, differentiation, and prolife- kinase I to the -catenin degradation complex and ration. A unique activity of SF/HGF and c-met on epithelial acts in both canonical Wnt and Wnt/JNK signaling cells in culture is the ability to induce branching or other mor- Thomas Schwarz-Romond, Christian Asbrand, Hans-Jörg phogenic events. We have identified a new substrate of c-met, Schaeffer, and Jörg Hülsken. In collaboration with Jürgen Gab1, which mediates the signal responsible for branching Behrens (Univ. Erlangen), Matthias Hammerschmidt and morphogenesis (Weidner et al., 1996; Schaeper et al., 2000; Jeroen Bakkers (MPI Freiburg), and Michael Kühl (Univ. Ulm). Sachs et al., 2000). Gab1 is a member of the family of membrane-bound multiadapter proteins, which transmits signaling An alternative branch of the Wnt pathway uses JNK to of tyrosine kinase receptors (C. Birchmeier, et al., 2003). establish planar cell polarity in Drosophila and gastrulation 67

We have analyzed the interactions between Wnt/-catenin and BMP receptor signalling during limb development using conditional mutagenesis, which allowed us to introduce loss-of-function (-catflox) and gain-of-function (N-catflox) mutations of -catenin, the central and essential mediator of canonical Wnt signalling. In addition, we generated compound mutant mice that carry both a gain-of-function mutation in -catenin and loss-of-function mutations in Bmp receptor IA (N-catflox; BmpRIAflox). Our analysis of these compound Brn4Cre; N--cateninflox; BmpRIAflox/flox mutant mice demonstrates that -catenin acts downstream of the BMP receptor IA in the induction of the proximal-distal axis (AER, apical ectodermal ridge). In contrast, our data suggest that -catenin acts upstream or in parallel to the BMP receptor IA during dorsal-ventral patterning. The intricate interactions between the Wnt/-catenin and BMP-signaling pathways provide the molecular basis that connects the development of proximal-distal and dorsal-ventral axes in the limb, and might thus ensure a tight spatial-temporal control of signalling responses.

Figure A) -Catenin acts downstream of the BMP receptor IA during AER formation Figure B) -Catenin acts upstream of the BMP receptor IA during dorsal-ventral (see scheme on the right). (A-D) AER is absent or only few groups of cells resembling patterning of limbs (see scheme at right). (A,C) En-1 is not expressed in ventral limb AER are present in -catenin loss-of-function mutant limbs at the 42 somite stage. (E,F) ectoderm of mutants carrying loss-of-function mutation of -catenin at the 30 somite AER and overall size of limb are strongly enlarged in -catenin gain-of-function mutants stage. (B,D) Wnt-7a is expressed ectopically in ventral ectoderm of -catenin loss-of- at the 42 somite stage. (G,H) AER is not formed in Bmp receptor IA loss-of-function function mutant limbs at the 30-somite stage. (E,F) En-1 and Wnt-7a expression mutants at the 42 somite stage. (I,J) Limbs are strongly enlarged and the AER is domains in -catenin gain-of-function mutants resemble the wild type. (G) En-1 is not expanded to ventral side in compound mutants at the 42 somite stage (cf. E,F). Dorsal- expressed in ventral ectoderm of mutants carrying loss-of-function mutations of Bmp ventral is as indicated. Bar, 100 µm. receptor IA. (H) Wnt-7a is expressed in both dorsal and ventral ectoderm in mutants carrying loss-of-function mutation of Bmp receptor IA. (I) En-1 is not expressed in the ventral ectoderm of compound mutants at the 30 somite stage. (J) Wnt-7a is expressed in ventral limb ectoderm of compound mutants at the 30 somite stage (cf. H). Dorsal ventral is as indicated. Bar, 50 µm.

movements in vertebrates. We have identified the novel verte- -Catenin regulates Cripto and Wnt3-dependent brate protein Diversin that interacts with two components of gene expression programs in mouse axis and the canonical Wnt pathway, Casein kinase I (CKI) and mesoderm formation Axin/Conductin. Diversion recruits CKI to the -catenin Markus Morkel and Jörg Hülsken. In collaboration with Maki degradation complex that consists of Axin/Conductin and Wakamiya and Richard Behringer (Univ. Texas, Houston), GSK3 and allows efficient phosphorylation of -catenin, Jixiang Ding and Michael Shen (UMDNJ Piscataway), thereby inhibiting -catenin/Tcf signals. Morpholino-based Makoto Taketo (Univ. Kyoto) and Marc van de Wetering and gene ablation in zebrafish shows that Diversin is crucial for Hans Clevers (Univ. Utrecht). axis formation, which depends on -catenin signaling. Diver- sin is also involved in JNK activation and gastrulation move- Gene expression profiling (Affimetrix) of -catenin, Cripto, ments in zebrafish. Diversin is distantly related to Diego of and Wnt3 mutant mouse embryos has been used to characte- Drosophila that functions only in the pathway that controls rize the genetic networks that regulate early embryonic deve- planer cell polarity. Our data show that Diversin is an essential lopment. We have defined genes whose expression is regula- component of the Wnt-signaling pathway and acts as a mole- ted by -catenin during formation of the antero-posterior axis cular switch that suppresses Wnt signals mediated by the and the mesoderm and have identified Cripto, which encodes canonical -catenin pathway and stimulates signaling via a Nodal co-receptor, as a primary target of -catenin signals JNK. both in embryogenesis as well as in colon carcinoma cell lines and tissues. We have also defined groups of genes that are regulated by Wnt3/-catenin signaling during primitive streak and mesoderm formation. Our data assign a key role to -catenin upstream to two distinct gene expression programs during antero-posterior axis and mesoderm formation. 68

Selected Publications Thomas Schwarz-Romond* Natalia Sochnikova Birchmeier, C., W. Birchmeier, E. Gherardi, and Vande Katja Großmann Woude, G.F. (2003): Met, Metastasis, Motility and more. Alexandra Klaus* Nature Reviews Molecular Cell Biology 4, 915-925. Heinz Möller*

Morkel, M., J. Huelsken, M. Wakamiya, J. Ding, M. van de Technical Assistants Wetering, H. Clevers, M.M. Taketo, R.R. Behringer, M.M. Katharina Feller* Shen, and Birchmeier, W. (2003). Beta-catenin regulates Renate Franke Cripto- and Wnt3-dependent gene expression programs in Frauke Kosel mouse axis and mesoderm formation. Development 130, Regina Vogel 6283-6294. Sabine Wilhelm Ingrid Walther Soshnikova, N., D. Zechner, J. Huelsken, Y. Mishina, R.R. Behringer, M.M. Taketo, E.B. Crenshaw, and Birchmeier, W Secretariat (2003): Genetic interaction between Wnt/beta-catenin and Irmgard Wiznerowicz BMP receptor signaling during formation of the AER and the Gerhild Richter dorsal-ventral axis in the limb. Genes & Development 17:1963-1968. * part of the time reported

Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Molina Leddy, H.E., Behrens, J., Sommer, T., and Birchmeier, W. (2002): Hakai, a novel c-cbl-like protein, ubiquitinates and induces endocytosis of the E-Cadherin complex. Nature Cell Biol., 4, 222-231.

Schwarz-Romond, T., Asbrand, C., Bakkers, J., Kühl, M., Schaeffer, H.-J., Huelsken, J., Hammerschmidt, M., and Birchmeier, W. (2002): The ankyrin repaet protein Diversin recruits Casein kinase Ie to the beta-catenin degradation complex and acts in both canonical Wnt and Wnt/JNK signaling. Genes & Development 16, 2073-2084.

Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G., and Birchmeier, W. (2001): Beta-catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105, 533-545.

Structure of the Group

Group Leader Prof. Dr. Walter Birchmeier

Scientists Dr. Felix Brembeck Dr. Yasuyuki Fujita* Dr. Jörg Hülsken* Dr. Jens-Peter von Kries* Dr. Li Li* Dr. Markus Morkel Dr. Barbara Munz* Dr. Marta Rosário Dr. Hans-Jörg Schaeffer Dr. Ute Schaeper Dr. Dietmar Zechner*

Graduate Students Christian Asbrand* Jolanta Chmielowiec* Boris Jerchow* Gunnar Schütz 69

Genetics of Tumor Progression and migration and invasion assays. In the future, we would like to Metastasis understand how these molecules regulate tumor progression and metastasis.

Ulrike Ziebold (Helmholtz Fellow) Mechanisms of tumorigenesis in murine embryonic stem cells

We will embark on aiding the detection of tumors by searching for novel marker genes and mechanisms for tumor initiation using murine embryonic stem cells (mES). Undifferen- tiated mES have the potential to grow tumors if transplanted into hosts. It is thus our hypothesis that the transition phase from the undifferentiated to the differentiated state is the focal point of tumor-suppressor and proto-oncogene action. We are developing a gene-trap screen to molecularly dissect the phenotypic changes of this transition phase by monitoring all transcriptional changes. Mutant mES-lines of our screen enable us to directly assess in vivo functions of promising candidates in the mouse. Ultimately, we wish to test the hypothesis if there are molecules and mechanisms that innately connect the control of stem cell proliferation and differentiation with Our laboratory concentrates on the unraveling of mechanisms the initiation and progression of tumors. underlying tumor formation, progression, and metastasis. It is well established that the change of a normal cell into a metastatic cancer cell arises due to the accumulation of mutations Selected Publications in proto-oncogenes and tumorsuppressors. In our group, we would like to understand the consequences of these molecular Ziebold, U., Caron, A., Bronson, R. and Lees, J.A. (2003). defects. Therefore, we will rely first on established and newly E2F3-loss has opposing effects on different pRb-deficient created mouse cancer models and, secondly, on genetically tumors, resulting in suppression of pituitary tumors but meta- modified murine embryonic stem cells (mES). stasis of medullary thyroid carcinomas. Mol.Cell.Biol. 18; 6542-6552.

The nature of the interaction of pRB and E2F Lee, E.Y., Cam, H., Ziebold, U., Rayman, J.B., Lees, J.A. and Dynlacht, B.D. (2002). E2F4 Loss suppresses Tumorigenesis The retinoblastoma protein (pRB), one of the first tumor in RB Mutant Mice Through a Novel Mechanism. Cancer suppressor proteins identified, is known to initiate tumors in Cell; 2; 463-472. both humans and mice. Recently, we have shown that E2F3, one member of the E2F-family, is a key downstream target of Cloud, J.E., Rogers, C., Reza, T.L., Ziebold, U., Stone, J.R., pRB. Surprisingly, E2F3-loss was able to promote or suppress Picard, M.H., Caron, A.M., Bronson, R.T. and Lees, J.A. the development of specific tumors. This demonstrated for (2002). Mutant mouse models reveal the relative roles of the first time that, in the absence of Rb, E2F3 possesses tumor E2F1 and E2F3 in vivo. Mol.Cell. Biol. 22(8); 2663-2672. suppressive functions. Currently, we are analyzing this tumor suppressive function of E2F3 in the development of Ziebold, U., Reza, T., Caron, A. & Lees, J.A. (2001). E2F3 mammary tumors. We also established novel E2F3 inducible contributes both to the proliferation and to the apoptosis knock-in mice. Ultimately, we wish to uncover the full bio- arising in Rb mutant embryos. Genes Dev., 15(4); 386-391. logical tumor functions of E2F3 and pRB. Perren, A., Weng, L.P., Boag, A.H., Ziebold, U., Thakore, K., Dahia, P.L., Komminoth, P., Lees, J.A., Mulligan, L.M., Finding novel molecules that regulate progression Mutter, G.L. & Eng, C. (1999). Immunohistochemical of tumors and metastasis evidence of loss of PTEN expression in primary ductal adeno- In collaboration with M. Morkel, J. Fritzmann, W. Birchmeier carcinomas of the breast. Am. J. Pathol. 155 (4); 1253-1260. and P. Schlag, at the MDC and the Robert-Rössle-Klinik/ Charité. Structure of the Group Using Rb/E2f3 mutant mice that develop aggressive mouse medullary thyroid carcinomas (MTCs), which metastasize to Group Leader numerous organs, we hope to gain new insight into the nature Dr. Ulrike S. Ziebold of common and distinct regulators of the onset of metastasis. Currently, we are using “micro-array gene-chip” analysis of Graduate Student Technical Assistant staged mouse and human tumors for comparative analysis. Caroline Boden Susanne Lützkendorf Potential candidates of our array will be tested in functional 70

Surgical Oncology A newly identified gene 7a5 is of prognostic value for metastasis of human colon carcinomas U. Stein, W. Walther, H. Schwabe, F. Arlt, P.M. Schlag. In Peter M. Schlag cooperation with W. Birchmeier, MDC, I. Petersen, Institute of Pathology, Charité, I. Fichtner, MDC, and H. Kalthoff, University of Kiel

We identified a novel gene, preliminarily referred to as 7a5, by comparing the gene expression patterns in human primary tumors, metastases of different target organs, and in mucosa of colon cancer patients. We identified the full-length cDNA sequence of 2559 bp by using EST cluster analysis. The encoded putative protein of 852 amino acids harbors defined domains, such as a src-homology-domain (SH3)-binding motif for interaction with other proteins, and potential tyrosine phosphorylation sites. The gene 7a5 is localized on the human chromosome 7. Stably 7a5-transduced clones were generated and showed higher migration behavior with respect to their 7a5-overexpression. In order to investigate the impact of 7a5 on in vivo tumor growth, stably 7a5-transduced tumor cell clones were subcutaneously applied in nude mice. These S100A4/metastasin expression depends on mutat- experiments revealed an increased growth of the 7a5-over- ed or wild-type beta-catenin in knock-out strains expressing tumors. After orthotopic transplantation, highest U. Stein, W. Walther, P.M. Schlag. In cooperation with E.D. tumors volumes and masses were again determined in those Harris, S.D. Mertins, and R.H. Shoemaker, National Cancer tumors showing the highest expression of 7a5. In human Institute, Frederick, MD, and T. Waldman, Georgetown Uni- colon carcinomas and their metastases, 7a5 expression was versity, Washington, DC detected by in situ-hybridization, real time RT-PCR as well as by immunohistochemistry using a newly generated poly- Beta-catenin has been implicated as an oncoprotein in colon clonal antibody. Furthermore, we examined microdissected and other cancer types. The effects of individual mutations surgical specimens of more than 70 patients with non-meta- and their potential role in the malignant phenotype are diffi- stasizing and metastasizing primary tumors, their metastases cult to assess. In order to isolate the effect of beta-catenin- of different target organs, as well as the corresponding normal mutations, gene knock-out technology was used to create iso- tissues. 7a5 expression levels are significantly increased in genic strains of the HCT116 human tumor cell line which the malignant tissues when compared to the corresponding express only one wild-type or one mutant allele of this gene. normal tissues and were found to be higher in the metastases We have coupled this to DNA array technology to identify versus primary tumors. More importantly, 7a5 expression those genes whose expression is dependent on beta-catenin levels were measured to be significantly lower in those genotype. cDNA array analysis of the human colon carcinoma primary tumors which did not metastasize, neither syn- nor cell line HCT116 in comparison to a derived knock-out strain metachronously (time observed: up to 120 month), versus tu- expressing only wild-type beta-catenin indicated dramatic mors which developed metastases within the next 120 forty-fold down-regulation of expression of the gene S100A4 months. These results suggest a prognostic role for 7a5 in (metastasin), known to be associated with the metastatic primary colon tumors in order to predict the probability of phenotype. This effect was confirmed by quantitative real developing distant metastases. time RT-PCR and evaluation of protein levels, and by extended study of additional knock-out strains and a naturally nullosomic tumor cell line NCI-H28. In NCI-H28 clones Hyperthermia and response-associated genes in stably transduced with the mutated beta-catenin, S100A4 human tumors mRNA levels were increased up to 70-fold, and in clones U. Stein, P. Hohenberger, W. Walther, P.M. Schlag. In coope- harboring the wild type beta-catenin exclusively, S100A4 ration with K. Jürchott, H. Lage and M. Dietel, Institute of expression was enhanced approximately 10-fold. In vitro Pathology, Charité, S. Bates, National Cancer Institute, migration was enhanced in clones stably transduced with the Bethesda, MD and T. Litman, University of Copenhagen mutated beta-catenin. EMSA showed binding of both the beta-catenin/TCF-4 complex and of TCF-4 to the S100A4 Isolated, hyperthermic limb perfusion (ILP) with rhTNF- promoter region in HCT116 cells, but only the binding of (TNF) and melphalan is a highly effective treatment for TCF-4 was detectable in NCI-H28 cells. Promoter activity of advanced soft tissue sarcoma (STS) and locoregional metasta- different deletion mutants of the S100A4 promoter was de- tic malignant melanoma. Since multidrug resistance (MDR)- pendent on the presence of the TCF-4 consensus sequence. associated genes are known to be inducible by heat and drugs, Comparison of the S100A4 status in the wild-type and mutant expression levels of the MVP, MDR1, and MRP1 were deter- knock-outs with the parental heterozygous cell line HCT116 mined sequentially before, during, and after ILP. STS or and with NCI-H28 cells suggests that mutant beta-catenin malignant melanoma patients were treated by ILP and tumor drives expression of S100A4 and does so in a dominant tissue temperatures were recorded continuously ranging from fashion when present in the heterozygous state. 33.4°C initially to peak values of 40.4°C during ILP. 71

most cases, more than five years. However, even in a group of patients with such a favorable prognosis, several of the patients die early due to post-operative formation of metastases. The aim of the present study was to identify genes which will allow detection of such high-risk patients. Expression profiles of colonic carcinomas were studied by oligonucleotide arrays using a novel strategy. Gene expression profiles of early staged colonic carcinomas from patients with a good survival were compared with those from patients with a poor prognosis. Since we suspect that normal mucosa of tumor patients is not as normal as expected, colonic mucosa of healthy individuals was taken as a reference point. In each case, colonic epithelium was captured using laser-micro- dissection. Processed and labeled RNA was hybridized to Affymetrix GeneChips U95A, U95B, U95C, U95D, and Intracellular distribution of jet-injected naked rhodamine-labeled plasmid-DNA in xeno- U95E containing about 60,000 sequences, most of them transplanted human colon carcinoma tissue. The confocal laser scanning microscopy ESTs. No additional amplification of the RNA was perfor- shows the plasmid-DNA (red) in close association to the nuclei (blue) of tumor cells within the tissue 30 minutes after jet-injection. med, in order not to perturb the original representation of the RNA-sequences. Only sequences with a more than 4-fold differential expression difference were further examined. GeneChip analysis led to the identification of a number of candidate genes which show a strong deregulated expression between epithelial cells of healthy and tumor patients. More- We found in 83% of the patients, that the expression of MVP over, several genes showing a strong overexpression in the was induced during hyperthermic ILP at the mRNA as well as carcinoma cells of patients with poor survival could be iden- at the protein level. Elevated MVP protein expressions were tified. Evaluation of the candidate gene expression in about observed either simultaneously with the MVP-mRNA induc- 60 cases of colorectal carcinomas with a well-documented tion, or timely delayed, following the induction at the follow-up by quantitative Taqman RT-PCR supports the transcriptional level. MVP-mRNA inductions often paralleled results found by GeneChip analysis. Tissue-specific expres- the increase in temperature during ILP. These temperatures sion of the putative marker genes was also evaluated by in- and the drugs applied preferably led to an induction of MVP situ hybridization and in some cases by immunhistochemi- and were not sufficient to induce MDR1 and MRP1 in the stry. Meanwhile, 90 candidate genes arisen from the majority of tumors. This study is the first to analyze the GeneChip experiments have been spotted onto glass slides. expression of MDR-associated genes sequentially during ILP The feasibility of such a OncoChip comprising a definite set of patients and demonstrates that treatment might lead to of putative marker genes for prognosis and diagnosis of colon elevated levels of MVP, whereas enhanced levels of MDR1 cancers is now under examination. and MRP1 remain rare events in the sarcomas and melanomas analyzed. Non-viral cancer gene therapy using jet-injection Moreover, a panel of thermoresistant and/or chemoresistant W. Walther, U. Stein, R. Siegel, P.M. Schlag. In cooperation human gastric carcinoma sublines, which were continously with EMS Medical GmbH, Nyon, Schweiz, and Plasmid grown at elevated temperatures, was analyzed in order to Factory, Bielefeld evaluate the impact of ABC-transporters such as BCRP, MRP1, and MDR1 on thermoresistance. Expression of the Various procedures are employed to deliver naked DNA into ABC-transporters was found to be dependent on the classical the desired cells or tissues in vivo. Among the various non-vi- or atypical type of chemoresistance. Furthermore, expression ral gene delivery technologies jet-injection is gaining increas- of the MDR-related ABC-transporters was increased in all ing acceptance, since this technique allows efficient gene thermoresistant counterparts, relative to the thermosensitive transfer into different tissues. In cooperation with EMS Medi- sublines, and overexpressed ABC-transporters were shown to cal a jet-injector prototype was created and tested for efficient be functionally active due to this long-term hyperthermic in vivo gene transfer. The key parameters of in vivo jet-injec- condition. tion, such as jet-injection volume, pressure, jet-penetration into the tumor tissue, DNA stability, and bio-distribution of jet-injected naked DNA, have been analyzed for optimized Expression profiling of early staged colon non-viral gene therapy. Therapeutic in vivo experiments carcinomas using the jet-injection transfer of the cytosine deaminase W. Kemmner, C. Astrosini, W. Haensch, P.M. Schlag. In (CD) suicide gene demonstrated antitumor effects. In these in cooperation with H.J. Gabius¸ Ludwig-Maximilians-University vivo studies, human colon carcinoma bearing NMRI-nu/nu Munich, M. Höcker, Charitè, A. Poustka, DKFZ, Heidelberg, mice were jet-injected with the CD gene harboring vector and J. Reich, MDC, H. Okamoto, Tokohu University, Japan. plasmid. Starting from day four of 5-fluorocytosine treatment, antitumor effects were seen in the CD-gene transduced Survival of patients with early staged colorectal carcinomas tumors compared to the non-jet-injected control group. The showing neither lymph node nor distant metastases, is, in growth inhibitory effect lasted for the entire observation time 72

of 24 days and showed significant growth inhibition of the Structure of the Group jet-injected colon carcinomas compared to the non-transduced but 5-fluorouracil treated animals. Group Leader Prof. Dr. Peter M. Schlag Based on our in vivo experiments, a phase I-study is planned to evaluate the feasibility of jet-injection aided LacZ reporter Scientists gene transfer in human colon- and mammary tumors. The Dr. Georgi Graschew results of this trial will provide the basis for the use of jet- Dr. Wolfgang Kemmner injection to apply therapeutic genes for the treatment of Dr. Wolfgang Haensch patients with locally advanced colorectal cancer. These genes Dr. Barbara Lustig will be regulated by hyperthermia-inducible vectors to Dr. Ulrike Stein combine gene therapy with hyperthermia. Dr. Wolfgang Walther

Graduate and Undergraduate Students Selected Publications Franziska Arlt Christian Astrosini Schaefer G, Cramer T, Suske G, Kemmner W, Wiedenmann Stephan Bergmann B, Hoecker M. (2003) Oxidative stress regulates vascular Bettina Georg endothelial growth factor-A gene transcription through Sp1- Holger Schwabe and Sp3-dependent activation of two proximal GC-rich pro- Shaoqiang Lin moter elements. J Biol Chem 278:8190-8198. Technical Assistants Walther W, Stein U, Voß C, Schmidt T, Schleef M, Schlag Jutta Aumann PM. (2003) Stability analysis for long term storage of naked Janice Smith DNA: impact on non-viral in vivo gene transfer. Anal Bio- Sabine Grigull chem 318:230-235. Claudia Röefzaad

Lustig B, Jerchow B, Sachs M, Weiler S, Pietsch T, Karsten U, van de Wetering M, Clevers H, Schlag PM, Birchmeier W, Behrens J. (2002) Negative feedback loop of Wnt signaling through upregulation of conductin/axin2 in colorectal and liver tumors. Mol Cell Biol 22:1184-1193.

Stein U, Jürchott K, Schläfke M, Hohenberger P. (2002) Expression of multidrug resistance genes MVP, MDR1, and MRP1 determined sequentially before, during, and after hyperthermic isolated limb perfusion of soft tissue sarcoma and melanoma patients. J Clin Oncol 20:3282-3292.

Walther W, Stein U, Schlag PM. (2002) Use of the human mdr1 promoter for heat-inducible expression of therapeutic genes.Int J Cancer 98:291-296.

Stein U, Lage H, Jordan A, Walther W, Bates SE, Litman T, Hohenberger P, Dietel M. (2002) Impact of BCRP/MXR, MRP1, and MDR1/P-glycoprotein on thermoresistant variants of atypical and classical multidrug resistant cancer cells. Int J Cancer 97:751-760. 73

Molecular Genetics of Cell action, both transcription factors instruct myeloid gene Differentiation & Tumorigenesis expression, even in fibroblasts. Mutations in either transcription factor may abrogate their collaboration, disrupt the myeloid differentiation program, and contribute to leukemia. Achim Leutz This concept has meanwhile been extended to several other co-operating hematopoietic transcription factors in different cell lineages.

Chromatin remodeling and lineage specific gene expression

A prerequisite for gene activation is to overcome the repressive effects of chromatin and to instruct polymerase II for transcription. C/EBP and - interact with the chromatin remodeling SWI-/-SNF complex and with ‘Mediator’, a complex that bridges transcription factors with the basic transcription machinery. The interaction between SWI/-SNF and C/EBP is required to modify chromatin and to activate silent differentiation genes in concert with other transcription factors, such as Myb in the hematopoietic system or PPAR in adipogenesis. Moreover, interaction with SWI/SNF is also Introduction required for C/EBP mediated proliferation arrest. As C/EBPs participate in many cell specification events, recruit- Hematopoietic stem cells in the bone marrow continuously ment of SWI/SNF may represent a major determinant of cell generate terminally differentiated blood cells of many diffe- lineage commitment and terminal differentiation. rent cell lineages such as erythrocytes, granulocytes, or macrophages. In addition, hematopoietic stem cells sustain their own maintenance - a condition called self-renewal. De-regulated cell differentiation or defective control of self-renewal may cause various diseases such as immune defects or leukemia. Thus, hematopoiesis provides striking examples to address fundamental biological and clinically relevant questions related to self-renewal of stem cells, cell lineage commitment, restricted progenitor proliferation, and cell Schematic representation of hematopoiesis. differentiation control. Hematopoietic stem cells in the bone marrow can either self-renew (gray arrow on the right) or give rise to progenitor cells that generate precursors of the myeloid or the lymphoid lineage. The commitment process is characterized by massive cell proliferation in the early phase followed by successive restriction to distinct cell lineages and to Combinatorial control of gene expression cell differentiation. These processes are regulated by trans-acting factors which activate or repress genes and lay down epigenetic changes in the chromatin of cells. This way, cells remember what they are, where they came from, and where they need to go. Cell development and differentiation programs are accom- Leukemic mutations interfere with transcription factor functions, abrogate cell differentiation, and support proliferation. As a consequence, the blood is flooded with imma- plished by switching on and off distinct sets of genes. Gene ture, non-functional cell types. regulatory proteins (transcription factors) that are downstream of signaling cascades bind to control regions of deve- lopmentally important genes and suppress or activate their expression. Specificity is primarily achieved by combinatorial control, i.e., through physical and functional interactions between several transcription factors that are simultaneously required at the same target genes. Combinatorial gene swit- ches permit plasticity of regulation and allow a multitude of developmental decisions with a limited number of regulators. Many of such important gene regulatory proteins, however, are also prone to tumorigenic conversion by mutations.

Several years ago, we identified the first combinatorial molecular switch that instructs cells to express myeloid genes. The switch consists of two types of transcription factors that are also involved in leukemogenesis, namely: 1) proteins of the CCAAT-/Enhancer Binding Protein family (C/EBP) regulate differentiation and cell cycle arrest and 2) the product of the Myb proto-oncogene that is essential for the development and maintenance of all hematopoietic lineages. In a concerted 74

Mediator: A connection between Ras signaling and regulatory complexes. These proteins provide “missing links” C/EBPb activation in understanding the complexity of gene regulation. We are therefore searching for proteins that interact with hemato- C/EBP is an intrinsically inhibited transcription factor that poietic transcription factors and oncoproteins using the acts as a repressor in the absence of signaling and that is “yeast-two-hybrid system” or by protein purification affinity turned into an activator by the Ras oncoprotein. C/EBP is protocols. A number of interesting proteins have already been phosphorylated through the Ras/MAP-kinase pathway and identified, e.g., proteins that harbor domains implicated in this phosphorylation event is accompanied by a conformatio- chromatin regulation during development or proteins with nal change of C/EBP. We found that active and repressive catalytic activity implied in the posttranslational modification C/EBP interacts with two different types of evolutionary of the transcription apparatus. We are developing murine conserved multi-subunit complexes that have been termed knockouts and knock-ins as well as RNAi strategies to deter- “Mediator” and that connect transcription factors with the mine the effects of C/EBP mutants that are defective for basic transcription machinery, including polymerase II. In its interactions with several of these co-factors. repressive form, C/EBP preferentially binds to repressive Mediator whereas oncogenic Ras signaling selects the transcriptionally active Mediator complex that also associates Selected Publications with RNA polymerase II. This suggests that a Ras-induced structural alteration of C/EBP determines differential gene Mo X., Kowenz-Leutz E., Xu H., and Leutz A. (2004) Ras activation through selective interaction with distinct Mediator Induces Mediator Complex Exchange on C/EBP . Molecular complexes. Cell: 13:1-10

Calkhoven C. F., Müller C., Martin R., Krosl G., Pietsch H., Translational regulation of transcription factors Hoang T., and Leutz A. (2003) Translational control of SCL isoform expression in hematopoietic lineage choice. Genes & From several hematopoietic transcription factor mRNAs, dif- Development, 17:959-964 ferent protein isoforms arise by initiation of translation at alternative start sites. The resulting transcription factor iso- Pedersen T.A, Kowenz-Leutz E., Leutz A. and Nerlov C. forms harbor distinct N-termini domains that may organize (shared first and senior authorships) (2001) Cooperation bet- different co-factor complexes with distinct functions in gene ween C/EBP TBP/TFIIB and SWI/SNF recruiting domains regulation. Hence, regulation of translation initiation may is required for adipocyte differentiation. Genes & Develop- play a crucial role in the control of cell fate. We have shown ment, 15:3208-3216 that this is the case with C/EBP, -, and the Stem Cell and T-Cell Leukemia Transcription Factor, and Scl/Tal1, Distinct Calkhoven, C.F., Müller, C., and Leutz, A. (2000) Translatio- isoforms of these transcription factors display specific func- nal control of C/EBP alpha and C/EBP beta isoform expres- tions in proliferation control, activation of genes, and sion. Genes & Development, 14: 1920-1932 differentiation. These findings suggest an important role of translation initiation control in hematopoiesis and leukemo- Kowenz-Leutz, E. and Leutz, A. (1999) A C/EBP-beta Iso- genesis. form Recruits the SWI/SNF Complex to Activate Myeloid Genes. Molecular Cell, 4: 735-743 Alternative initiation of translation may be prompted by small upstream open reading frames (uORF) that are located in the 5’ region of the respective mRNAs. Such uORFs perceive the Structure of the Group activity of the translation initiation machinery that is sensitive to environmental inputs such as stress, nutrition, or hormonal Group Leader Technical Assistants changes. This ancient mechanism of gene regulation is Dr. Achim Leutz Nadine Burbach already found in yeast and allows cells to rapidly adjust to Carola Geiler environmental changes. Proteins involved in translational Scientists Maria Knoblich control include PI3-kinase, AKT-kinase, PTEN-phosphatase Dr. Valerie Begay or translation initiation factors eIF-2 or eIF-4E, many of Dr. Elisabeth Kowenz-Leutz Secretariat which can turn into oncogenes. It is anticipated that pathways Dr. Xianming Mo Henriette Mödig and factors involved in the control of translational initiation Dr. Christine Müller* may play a far more important role than previously recog- Dr. Marina Scheller Associated Helmholtz-Group nized and that they represent novel targets for innovative drug Dr. Jeske Smink Dr. Cor Calkhoven therapies. Accordingly, we are developing screening systems Volker Wiesenthal to discover appropriate drugs. Graduate Students Christiane Calließ Paula Babarovic Simone Joschko* Transcription co-factors Ole Pless Xiaojin Sha Transcription factors interact with other transregulatory pro- Hong Xu teins that function as co-activators, co-repressors, chromatin Katrin Zaragoza remodeling factors, and/-or bridging factors between gene * part of the time reported 75

Translational Control of Gene first time in vertebrate cells, that translationally regulated Expression expression of SCL and C/EBP protein isoforms determines cell fate. In the hematopoietic system, the ratio of SCL isoforms determines lineage outcome of primary bone Cornelis F. Calkhoven (Helmholtz Fellow) marrow cells into either erythrocytes or megakaryocytes. We also demonstrated that translational deregulation of C/EBP isoform expression results in disturbed adipocyte differentiation and cellular transformation. Hence, translational deregulation of C/EBP isoform expression may be implicated in the development of tumors and metabolic disorders. Similarly, deregulation of SCL translation might be involved in its oncogenic potential as displayed in T-cell leukemia.

We discovered small upstream-open-reading-frames (uORFs) in the c/ebp and scl mRNAs that serve as cis-regulatory elements controlling the site of translation initiation. By monitoring the activity of translation initiation factors (eIFs), they determine the ratio of protein isoform expression. In this way, signal transduction pathways, which converge on ribosome activities, are linked to cell fate.

Several proteins of the translational control signaling network Our work focuses on the expression of key-regulators in and machinery, as well as translationally controlled genes, are cellular differentiation, proliferation, and function at the implicated in oncogenic, neurological, inflammatory, and mRNA-translation level. Research in the field of translation metabolic disorders. It is anticipated that translational control control has progressed rapidly, revealing new regulatory in vertebrate development and disease will prove to be of mechanisms and constantly augmenting the list of translatio- greater importance than previously thought and that it may in- nally regulated genes. Recently, the etiologies of several clude targets for therapeutics. Therefore, we have created a human diseases were linked to mutations in genes of the Translational-Control-Reporter-System (TCRS) designed for translational control machinery, highlighting the significance the identification of such agents to aid in the development of of this regulatory mechanism. In addition, deregulation of novel therapeutic strategies in treating cancer and other translational control is associated with a wide range of human diseases. cancers. Novel therapeutic strategies are being developed that target translational control, a promising concept in the treatment of human diseases. Selected Publications

We study the translationally controlled expression of the Calkhoven, C.F., Müller, C., Martin, R., Krosl, G., Pietsch, transcription factors CCAAT/Enhancer Binding Protein H., Hoang, T. and Leutz, A. (2003). Translational control of (C/EBP) and - and Stem Cell Leukemia factor (SCL) SCL isoform expression in hematopoietic lineage choice. which are essential in differentiation programs of different Genes & Dev. 17, 959-964. systems including hematopoiesis. We have shown that various protein isoforms of C/EBP and SCL are expressed Calkhoven, C.F., Müller, C. and Leutz, A. (2002). Translatio- from alternative translation initiation sites. The isoforms have nal control of gene expression and disease. Trends in Mole- distinct and partially opposing functions in gene regulation, cular Medicine. 8, 577-583. differentiation, and proliferation control. We showed, for the

Upregulation of truncated (Tr) C/EBP isoform expression results in cellular transformation of differentiated 3T3-L1 adipocytes. Aberrantly high levels of Tr-C/EBP were caused by the upregulation of translation initiation factor eIF4E or eIF2 activities, or ectopic overexpression of Tr-C/EBP. 3T3-L1 cells underwent a differentiation protocol, were fixed and stained for lipid droplets with Oil Red O as described in Calkhoven et al. (2000). (Fl) full-length C/EBPa; (Tr) truncated C/EBPa. 76

Calkhoven, C.F., Müller, C. and Leutz, A. (2000). Translatio- nal control of C/EBPa and C/EBPb isoform expression. Genes & Dev. 14, 1920-1932.

Structure of the Group

Group Leader Dr. Cornelis F. Calkhoven

Graduate Student Volker Wiesenthal

Technical Assistant Christiane Calließ 77

Signal Transduction in Tumor Cells logues Bcl-3 and MAIL. As an intriguing feature of NF-B transcription factors, two of its subunits, p50 and p52, are produced as precursor proteins, p105 and p100, respectively, Claus Scheidereit which require proteolytic processing by the proteasome. Unprocessed p105 and p100 bind to other NF-B subunits and so act as cytoplasmic inhibitors.

The signal-activated IKK complex phosphorylates IB, , and at a conserved signal response domain and this sequence, containing also lysines for phosphorylation-dependent ubiquitin-conjugation, is sufficient to confer inducible degradation of the IBs. In addition to this “classical” activation pathway triggered by IKK-mediated destruction of small IBs, IKK controls the fate of the NF-B precursors as well. On stimulation with pro-inflammatory NF-B activating agents, such as tumor necrosis factor (TNF) or bacterial lipopolysaccharides (LPS), cellular p105 is phosphorylated by IKKs at two serine residues. To bind the IKK complex, p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecule TrCP, resulting in polyubi- The main interest of our laboratory is to understand how quitination and complete degradation by the proteasome. signal transduction processes are coupled to transcription. A p105-associated NF-B subunits, such as its own processing model system with wide physiological and medical relevance product p50, which is continuously formed by basal process- is nuclear factor kappaB (NF-B) and its co-regulators and ing of p105, are then liberated and are transported to the accessory proteins. A major goal of our research is to under- nucleus to affect gene expression. In contrast to the signal- stand the structures and mechanisms underlying gene regu- induced complete degradation of p105, the structurally lation by this complex system and its implications in disease related p100 molecule undergoes stimulus-dependent development. processing. We have shown that lymphotoxin and LPS, but not other activators of classical NF-B, such as TNF or IL-1, induce proteolytic maturation of cytoplasmic p100 to its Pathways and structures that regulate NF-B p52 product, which then migrates to the nucleus and binds to activity DNA. Most surprisingly, the processing reaction, which involves IKK-induced p100 polyubiquitination and partial The pleiotropic transcription regulator NF-B plays a central digestion by the proteasome, can only take place while p100 role in the inducible expression of a large number of genes is being synthesized at the ribosome, not after translation is which encode cytokines, surface receptors, adhesion mole- completed. cules, transcription factors, and other molecules controlling various immune functions as well as cell proliferation and programmed cell death. In its inactive latent form, NF-B is Requirement of NF-B for embryonic development kept in the cytoplasm by association with IB molecules, of hair follicles, eccrine glands, and formation of which inhibit nuclear translocation and DNA binding activity secondary lymphoid organs of NF-B. Stimulation of cells with a variety of agents, such as bacterial lipopolysaccharides, tumor necrosis factor , Gene knockout studies in mice have revealed that single (TNF) or interleukin-1 (IL-1), results in the proteolysis NF-B and IKK subunits are essential for various steps in of IB molecules and liberation of active NF-B into the immune responses and inflammation, but also for bone- nucleus. Induced IB proteolysis is triggered by IB phos- morphogenesis and keratinocyte differentiation. However, phorylation mediated by an IB kinase (IKK) complex, which due to embryonic lethality and functional redundancy bet- is activated by many NF-B stimulating pathways and con- ween subunits, other physiological functions of the NF-B sists of catalytic (IKK, IKK) and regulatory (IKK) com- system were inaccessible. With a conditional gene targeting ponents. approach, we have ubiquitously expressed an NF-B superrepressor, IBN, to broadly downmodulate NF-B activity in the entire organism or in single organs and tissues. Mice Differential regulation of NF-B activity by small with suppressed NF-B display macrophage dysfunction and IBs and precursor molecules the lack of secondary lymphoid organs like Peyer’s patches and peripheral lymph nodes. NF-B inhibition further causes The mammalian NF-B family consists of five members, severe defects in the early developmental steps of epidermal p50, p52, p65, c-Rel, and RelB. These proteins form hetero- appendices, including hair follicles and tear and sweat glands. and homodimers and are bound by the inhibitory cytoplasmic Normally, these structures display strong NF-B transcriptio- IB molecules IB, and , or by the nuclear IB homo- nal activity, as we have demonstrated with -galactosidase reporter mice. The epidermal phenotype is analogous to 78

LT-12 LT-R

IBN IBN

IKK,, IKK IKK,

NF-B IBN NF-B

Two major signaling pathways essential for organogenesis, which require NF-B activity (Schmidt-Ullrich, 2001, Development 128, 3843). A knock-in strategy, where the NF-B superrepressor IBN was expressed in mice, revealed that NF-B is required to confer Ectodysplasin signals for the embryonal development of hair follicles (HF), sweat glands (SG) and other epidermal structures, the lack of which establish the hypohydritic ectodermal dysplasia (HED) phenotype (left). NF-B is also required for transmitting lymphotoxin signals to induce formation of secondary lymphoid organs (peripheral lymph nodes, LN, and Peyer’s patches, PP) (right). Signaling molecules which upon inactivation cause similar defects in both pathways are indicated in red.

hypohidrotic (anhydrotic) ectodermal dysplasia (HED) in We have found that in addition to IKK/NF-B, malignant humans, and identical to phenotypes of eda, edar or crinkled cells of Hodgkin’s disease bear a second aberrantly activated mice. The eda and edar genes belong to the TNF family of transcription factor. AP-1 (activating protein 1), composed of ligands and receptors, respectively. Our data thus indicate that the c-Jun and JunB subunits, is strongly overexpressed. c-Jun NF-B is required in epidermal development for edar to and JunB overexpression is observed in the great majority of transmit eda signals. Currently, the crosstalk of NF-B with tumor cells of all patients tested with classical Hodgkin’s other signaling pathways during epidermal organogenesis is disease, to a lesser extent in anaplastic large cell lymphoma, being investigated. but not in other B or T cell malignancies. Further analysis revealed that AP-1 supports proliferation of Hodgkin cells. In fact, AP-1 cooperates with NF-B to co-stimulate expression Role of IKK/NF-B and AP-1 signaling cascades in of the cell-cycle regulator cyclin D2 and the lymphocyte Hodgkin’s disease homing receptor CCR7, which are all strongly expressed in Hodgkin cells. These data suggest an important role for AP- In collaboration with the research group of Bernd Dörken, we 1/c-Jun and for a cooperation between IKK/NF-B and AP-1 previously discovered an essential role of aberrant constitu- in Hodgkin lymphoma pathogenesis. tive NF-B activity in the viability of Hodgkin’s disease (HD) tumor cells. The NF-B/IB system is dysregulated in a cell- autonomous manner, generally involving a persistent activa- Selected Publications tion of the IKK complex. Constitutive NF-B blocks apoptosis and promotes proliferation and tumorigenicity of the Mordmueller, B., Krappmann, D., Esen, M., Wegener, E., and malignant cells. We have now undertaken a genome wide Scheidereit, C. (2003) Lymphotoxin and lipopolysaccharide determination of genes which are activated by constitutive induce NF-B-p52 generation by a co-translational mecha- IKK/NF-B in Hodgkin’s disease. NF-B accounts for the nism, EMBO Rep. 4, 82-87. high level expression of a large group of genes, many of which represent characteristic expression markers for the Tegethoff, S., Behlke, J., and Scheidereit, C. (2003) Tetra- malignant cells. Thus, NF-B drives expression of genes meric oligomerization of IKK is obligatory for IB kinase which regulate cell cycle progression, inhibit programmed complex activity and NF-B activation. Mol. Cell.Biol. 23, cell death, direct tropism, and migration of tumor cells, their 2029-2041. resistance to chemotherapeutic drugs and abundant cytokine production, indicating a central pathogenetic role of the Choi, M., Rolle, S., Wellner, M., Cardoso, C., Scheidereit, C., IKK/NF-B pathway. Luft, F.C. and Kettritz, R. (2003) Inhibition of NF-B by a 79

TAT-NEMO-binding domain peptide accelerates constitutive apoptosis and abrogates LPS-delayed neutrophil apoptosis. Blood, 102, 2259-2267.

Mathas, S. Krappmann, D. Anagnostopoulos, I., Hinz, M., Jundt, F., Bommert, K., Lallemand, D., Stein, H. Dörken, B. and Scheidereit, C. (2002) Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-B. EMBO J., 21, 4104- 4113.

Hinz, M., Lemke, P., Anagnostopoulos, I., Hacker, C., Krappmann, D., Mathas, S., Dorken, B., Zenke, M., Stein, H. and Scheidereit, C. (2002) NF-B dependent gene expression profiling of Hodgkin’s disease tumor cells, pathogenetic significance and link to constitutive STAT5a activity, J. Exp. Med., 196, 605-17.

Structure of the Group

Group Leader Dr. Claus Scheidereit

Scientists Dr. Annette Ahlers Dr. Mira Choi Dr. Amina El Jamali Dr. Michael Hinz* Dr. Eva Kärgel* Dr. Daniel Krappmann Dr. Stefan Mathas* Dr. Benjamin Mordmüller* Dr. Ruth Schmidt-Ullrich

Graduate and Undergraduate Students Meike Bröhmer Jan Ebert* Christian Freund Yoshiaki Sunami* Sebastian Tegethoff* Elmar Wegener

Technical Assistants Erika Scharschmidt* Sabine Jungmann Rudolf Dettmer Karin Ganzel

Secretariat Daniela Keyner

* part of the time reported 80

Antigen Receptor Signaling In an approach to gain mechanistic insights into the processes in Lymphocytes that govern activation of the IKK complex after T and B cell activation, we have investigated post-translational modifications of the critical regulator Bcl10. PKC or T cell receptor Daniel Krappmann (Helmholtz Fellow) (TCR)/CD28 signaling results in phosphorylation and subsequent down-regulation of Bcl10 protein levels, thereby attenuating NF-B transcriptional activity. Bcl10 degradation requires an intact caspase recruitment domain and is not observed after stimulation with TNF- or LPS. Bcl10 downregulation is not affected by proteasome inhibitors but is accompanied by transient localization to lysosomal vesicles, suggesting involvement of the lysosomal pathway rather than the proteasome. The HECT-domain ubiquitin ligases NEDD4 and Itch promote ubiquitination and degradation of Bcl10, thus down-modulating NF-B activation. Since CD3/CD28 induced activation of JNK is not affected by the decline of Bcl10, degradation of Bcl10 selectively terminates IKK/NF- B signaling in response to TCR stimulation. Together, these results suggest a novel mechanism of negative signaling in which TCR/PKC signaling initially activates Bcl10 but later promotes its degradation- a process that might help to prevent abnormal lymphocyte activation and induce tolerance. Antigen receptors on T or B lymphocytes are responsible for inducing opposite responses of immunity or tolerance. A number of positive and negative regulatory mechanisms have Selected Publications been described. In T cells, immunogenic activation requires TCR (T cell receptor) engagement by antigenic peptides and Scharschmidt, E., Wegener, E., Heissmeyer, V., Rao, A. and a second co-stimulatory signal, of which CD28 is the most Krappmann, D. (2004) Degradation of Bcl10 induced by T prominent. Optimal TCR/CD28 ligation initiates a series of cell activation negatively regulates NF-B signaling. Mol. signal transduction events which modulate the activity of Cell. Biol. (in press). several nuclear transcription factors including NF-B, AP-1, and NF-AT, ultimately leading to differentiation and prolife- Krappmann, D., Patke, A., Heissmeyer, V. and Scheidereit, C. ration. In contrast, weak T cell activation, e.g. by poorly (2001) B-cell receptor- and phorbol ester-induced NF-B and cross-linking peptides or the absence of co-stimulus, promo- c-Jun N-terminal kinase activation in B cells requires novel tes an uncoupling of the TCR and their downstream signaling protein kinase C's. Mol. Cell. Biol. 21, 6640-50. pathways and induces T cell tolerance. Paradoxically, the same antigen receptor triggers both outcomes. Resolving how one receptor elicits such opposing effects is crucial for deter- Structure of the Group mining how specific immune responses can be selectively switched on or off to counteract organ rejection or to treat au- Group Leader toimmune diseases. Dr. Daniel Krappmann

Antigenic stimulation of the IkB kinase (IKK) complex and Graduate Students subsequent activation of NF-B is essential for clonal expan- Andrea Oeckinghaus sion of naïve B and T cells. Using Jurkat T cells and gene Elmar Wegener target disruption in mice, novel PKC was identified as a central component coupling TCR proximal signaling events Technical Assistant to IKK/NF-B activation. By the use of pharmacological Erika Scharschmidt inhibitors as well as rescue experiments in PKC deficient cells, we could demonstrate that also in mouse pre B cell lines and in primary B cells PKCs are essential for mediating B cell receptor (BCR) induced NF-B activation. In contrast, NF-B activation by cytokines or pathogenic stimuli is independent of PKCs. This study provided clear evidence that conserved upstream pathways trigger IKK activation in response to BCR and TCR clustering in both lymphocyte populations. This conservation was confirmed by more recent studies in which Bcl10, MALT1, and CARMA1 were identified as common signaling components required for bridging PKCs to IKK activation in response to antigen receptor stimulation in T and B cells. 81

Intracellular Proteolysis CD4 during its maturation in the ER. This proteolysis is mediated by Vpu, an HIV-encoded N-terminally anchored membrane protein that interacts with CD4. In addition, Vpu binds Thomas Sommer the human F-box protein TrCP, which functions as a substrate recognition factor of the multisubunit ubiquitin ligase SCF. TrCP recognizes a specific degradation signal contai- ned in the cytoplasmic tail of Vpu. This signal is also found in other short-lived substrates of the human SCFTrCP, like -catenin and IB, and comprises two phophorylated serine residues. However, in contrast to -catenin and IB, binding of TrCP does not lead to proteolysis of Vpu but instead to degradation of the associated CD4. However, ubiquitination of CD4 has not been shown. An additional open question is whether the known membrane-bound components of the ubiquitin system also participate in this process or whether Vpu mediated turnover of CD4 represents a separate pathway that may also be mechanistically distinct from ERAD of endogenous substrates.

To address these open questions, we have reconstituted Vpu mediated turnover of CD4 in yeast. A reconstitution in this model organism offers the advantage that mutants in all The secretory pathway of eukaryotic cells harbors an elabo- ERAD components can be used. Reconstitution of the viral rate protein quality control system which prevents the accu- degradation mechanism for CD4 was possible by co-expres- mulation of misfolded or unassembled proteins in the secre- sion of only Vpu and human TrCP. Interestingly, this system tory pathway. This system is localized in the Endoplasmic is active in the absence of the membrane-bound ERAD com- Reticulum (ER). ER associated protein degradation (ERAD) ponents. Thus, the viral system seems to work independently is an important component of this quality assurance system of the endogenous ERAD pathway. In addition, we found that and directs misfolded proteins for destruction by the cyto- CD4 expressed in the absence of Vpu and TrCP is a target of plasmic ubiquitin-proteasome pathway. endogenous yeast ERAD. This unique situation allows a direct comparison of the viral and the cellular degradation ERAD can be divided mechanistically into separate steps: pathways for the same molecule. Our detailed analysis of the First, misfolded proteins are detected within the ER-lumen, a proteolysis of CD4 revealed mechanistic differences between step that, most likely, requires molecular chaperones. Second, the viral and the cellular pathway. Taken together, our results the proteolytic substrates are targeted to and inserted into an point to the fact that HIV hijacks an unrelated proteolytic aqueous transport channel that probably includes the multi- pathway, recruits it to the ER-surface, and uses it in degra- spanning membrane protein Sec61p. Third, the substrates are dation of CD4 from the membrane . transported back into the cytosol in a process termed dislocation or retro-translocation. Fourth, a polyubiquitin chain is synthesized on the dislocated substrates. This step requires Figure 1 the action of membrane-bound components of the ubiquitin The two pathways for degradation of the integral membrane protein CD4 are out- lined in this cartoon. system. In yeast, these are the ubiquitin-conjugating enzymes The viral mechanism involves the HIV encoded Vpu protein that binds to CD4. In addi- Ubc1p, Ubc6p, and Cue1p assembled Ubc7p and the ubi- tion, Vpu recruits the cytsolic ubiquitin ligase SCFTrCP to the ER-membrane, which attaches a polyubiquitin chain onto CD4. Degradation by the endogenous cellular ERAD quitin ligases Hrd1p and Doa10p. Fifth, the Cdc48p/Ufd1p/ system involves the classical ERAD components Hrd1p/Hrd3p and Ubc7p. Therefore, Npl4p ATPase complex mobilizes the ubiquitin-conjugated the viral mechanism is distinct from the cellular ERAD system. Furthermore, the viral mechanism depends on the presence of cytosolic lysine residues in the substrate as and dislocated molecules which are still attached to the cyto- acceptor sites for ubiquitin. In contrast, the endogenous ERAD system does not de- solic surface of the ER-membrane. Finally, the cytosolic 26S- pend on such cytosolic attachment sites, since it can transfer the lysine containing proteasome digests the ubiquitin-conjugated dislocated mole- luminal parts of CD4 into the cytoplasm. Thus, both pathways differ mechanistically: The viral system cannot transport parts of CD4 without prior ubiquitination, while the cules. cellular ERAD system is able to transport before ubiquitination takes place.

Our group has defined the components and the basic principles of ERAD in the last years. This process seems to occur in all eukaryotic organisms in a highly conserved manner. Mal- functions in this system lead to altered protein composition in the secretory pathway and may therefore cause serious human diseases like Cystic Fibrosis and Parkinson’s disease. More- over, it has been suggested that some human viruses co-opt the ERAD systems to destroy specific host proteins and manifest themselves in the infected cell.

One example of this is the human immunodeficiency virus (HIV) induced degradation of the plasma membrane protein 82

Selected Publications

Jarosch, E., Lenk, U., and Sommer, T. (2003) Endoplasmic reticulum-associated protein degradation. Int. Rev. Cyt., 223, 39-81.

Jarosch, E., Taxis, C., Volkwein, C., Bordallo, J., Finley, D., Wolf D. H., and Sommer, T. (2002) Protein Dislocation from the ER Requires Polyubiquitination and the AAA-ATPase Cdc48. Nature Cell Biol. 4, 134-139.

Fujita, Y., Krause, G., Scheffner, M., Zechner, D., Leddy, H.E., Behrens, J., Sommer, T., and Birchmeier, W. (2002) Hakai, a c-cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nature Cell Biol. 4, 222-231.

Lenk, U., Yu, H., Walter, J., Gelman, M., Hartmann, E., Kopito, R.R., and Sommer, T. (2002) A role for mammalian Ubc6 homologues in ER-associated protein degradation. J. Cell Science 115, 3007-3014.

Jarosch, E., Geiss-Friedlander, R., Meusser, B., Walter, J., and Sommer, T. (2002) Protein dislocation from the Endoplasmic Reticulum – Pulling out the suspect. Traffic 3, 530-536.

Sommer, T. and Jarosch, E. (2002) BiP binding keeps ATF6 at bay. Dev. Cell 3, 1-2

Structure of the Group

Group Leader Dr. Thomas Sommer

Scientists Dr. Ernst Jarosch Dr. Christian Hirsch

Graduate and Undergraduate Students Robert Gauss Ruth Geiss-Friedlander Birgit Meußer Oliver Neuber

Technical Assistants Corinna Volkwein Angelika Wittstruck

Secretariat Sylvia Klahn 83

Regulation of Nuclear Transport Selected Publications Processes Stade, K., Vogel, F., Schwienhorst, I., Meusser, B., Volkwein, C., Nentwig, B., Dohmen, R.J. and Sommer, T. (2002) A lack Katrin Stade (Helmholtz Fellow) of SUMO conjugation affects cNLS-dependent nuclear protein import in yeast J. Biol. Chem. 277, 49554-49561

Maurer, P., Redd, M. Solsbacher, J., Bischoff, F.R., Greiner, M., Podtelejnikov, V.P., Mann, M., Stade, K., Weis, K. and Schlenstedt, G. (2001) The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p) Mol. Biol. Cell, 12, 539-549

Stade, K., Ford, C.S., Guthrie, C. and Weis, K. (1997) Expor- tin1 (Crm1p) Is an Essential Nuclear Export Factor. Cell 90, 1041-1050

Structure of the Group

Group Leader In eukaryotic cells, genetic information is stored in the Dr. Katrin Stade nucleus, whereas protein synthesis occurs in the cytoplasm. This spatial separation of essential cellular processes, such as Graduate and Undergraduate Students transcription and translation, can only be overcome by allow- Anja Pannek ing macromolecules to traverse the nuclear membrane in both Sonja Schulz directions. Nucleocytoplasmic trafficking is therefore a major cellular activity, in terms of both number of individual particles involved and energy consumption. However, despite their enormous complexity, nuclear transport processes still offer an excellent opportunity for efficient regulation of cell cycle progression and signalling pathways. Our lab investigates a family of soluble nuclear transport receptors termed karyopherins which can be further classified as import and export receptors, so-called importins and exportins, respectively. Xpo1 was the first exportin to be described (1) and since then, detailed information has become available with respect to this particular transport receptor and the export pathway it is serving (2). However, much remains to be learned about Xpo1.

One of our current research interests is to identify Xpo1- specific export substrates and study their interaction with this exportin in more detail. In addition to nuclear protein export, a new reseach interest of the lab is the study of nuclear import processes and how they might be regulated by post-translational protein modification. In the past, protein phosphorylation was identified as an important means of regulating nuclear transport reactions. More recently, a rather novel protein modification system has been proposed to play a role in nucleocytoplasmic trafficking. The ubiquitin-like small modi- fier SUMO, which previously had been shown to play an important role in cell cycle progression, chromatin structure, and DNA repair, was also recognized as a key player for one particular nuclear protein import pathway in budding yeast (3). Ongoing studies in our lab now focus on the elucidation of SUMO’s exact role for nuclear transport reactions. 84

Initiation of DNA Replication Biochemical characterization of the human ORC

We were able to co-express genes for all six subunits of Manfred Gossen human ORC in insect cells and to purify the resulting protein complex to homogeneity. It turns out that human ORC is capable of forming various distinct sub-complexes, which differ in their stability and DNA binding properties. By omitt- ing individual subunits in this protocol, specific interactions among the ORC proteins could be revealed. According to the Saccharomyces cerevisiae paradigm, ORC’s binding to DNA is expected to be ATP-dependent. We are currently investigating this possibility by analyzing the subunits responsible for ATP binding as determined by nucleotide crosslinking and testing the biochemical properties of recombinant human ORC defective in ATP interactions. We will evaluate the significance of these findings for the role of ORC in DNA replication initiation by using an in vitro DNA replication protocol.

Ablation of MCM proteins

The research group is interested in the mechanisms controlling The genomes of all eukaryotic organisms code for six differ- the initiation of DNA replication in multicellular eukaryotes. ent MCM proteins which can interact with each other. Three In metazoans, the interplay between chromosomal cis ele- of them, MCM 4, 6, and 7, form the putative replicative DNA ments and the trans acting factors that contribute to the initia- helicase. The other subunits are believed to suppress or regu- tion of replication is poorly analysed. This, however, would be late the helicase activity. In a colloboration with atugen AG, a prerequisite for a detailed understanding of the processes Berlin, we are investigating the effects of special antisense controlling genome duplication and cellular proliferation. Cur- molecules (“Geneblocs”) directed against one of the MCM rent approaches in our laboratory include the analysis of pro- genes, Mcm4, in primary human fibroblasts. We have disco- teins forming the prereplicative complex (preRC)- specifi- vered that the knockdown of this gene results in the growth cally, members of the minichromosome maintenance (MCM) arrest of the cells, the stop of DNA synthesis, and an increase protein family and of the Origin Recognition Complex (ORC). in the G2/M population of the transfected cells. Interestingly, Currently, both Drosophila and mammalian tissue cultures are some other members of the MCM protein family are also effi- used as experimental systems. In addition, the group is inte- ciently down-regulated. The mechanisms involved in this rested in transcriptional cross-talk between neighboring trans- process are currently under investigation. genes and the long-term stability of their expression patterns.

Interference of closely spaced transcription units Localization of the Drosophila Origin Recognition Complex (ORC) Transcription units placed randomly in the chromosomes of mammalian cells are subject to both epigenetic control and ORC is likely to function as the initiator protein in eukaryotes the influence of nearby transcription signals. These findings (i. e. its binding to chromosomal sites specifies the origins of have important implications for the design of gene expression bidirectional DNA replication). These sites are only poorly vectors for transgenesis, and gene therapeutic approaches. characterized in metazoan organisms. Drosophila melano- Frequently, however, it is desirable to transfer more than one gaster offers several distinct advantages for the analysis of transcription unit in one step. We are analysing the effects replication initiation proteins. Among them are the availa- these transgenes exert on each other by using an inducible bility of a large number of hypomorphic alles of replication transcription system. Upon induction of a target gene, a initiation genes, an embryonic development which relies on neighboring, “constitutive” transcription unit can be co-regu- maternally supplied stockpiles of replication factors, and the lated depending on the nature of the promoters used. Vice finding that certain specialized origins in follicle cells can be versa, these promoters can have a dominant influence over the monitored individually by confocal microscopic techniques. characteristics of the inducible transcription unit. These To analyze the specificity of ORC DNA binding in vivo, we effects are furthermore analysed taking the effects of epigene- generated GFP fusion constructs with the gene for one of the tic transgene control like DNA methylation and chromatin subunits, Orc2, in its authentic chromosomal environment. compaction into account. This construct was expressed in transgenic flies in an Orc2- null background. This approach allows us to determine the subcellular localization of ORC throughout the cell cycle, in particular its chromatin association, and reveals changes in the dynamic behavior of this protein complex in different tissues and throughout development (see figure). 85

Drosophila ORC2 is excluded from metapahase chromosomes. A) Confocal microscopy of a 3 to 4 hour old rescue embryo (Orc2-GFP; Orc2 -/-). The Orc2-GFP signal is shown in green, DNA counterstain in red (only merged pictures are shown). Encircled in white is one of the mitotic domains, i. e. a patch of adjacent cells still progressing in synchrony through the cell cycle at this stage of embryonal development. B) The same mitotic domain (depicted is a neighboring Z-plane) in higher resolution. Note that there is no enrichment of the GFP signal on metapase chromosomes (white arrow), whereas in anaphase (white arrowhead) ORC2 is concentrated on the chromosome, with merged green and red channels resulting in the yellow signal.

Selected Publications

B Schories, K Engel, B Dörken, M Gossen, and K Bommert. (2004) Characterization of Apoptosis-induced Mcm3 and Cdc6 Cleavage Reveals a Pro-Apoptotic Effect for one Mcm3 Fragment. Cell Death & Differentiation, in press.

Fitze G, Appelt H, Konig IR, Gorgens H, Stein U, Walther W, Gossen M, Schreiber M, Ziegler A, Roesner D, Schackert HK. (2003) Functional haplotypes of the RET proto-oncogene promoter are associated with Hirschsprung disease (HSCR). Hum Mol Genet. 12(24):3207-14.

Gossen M, Bujard H. (2002) Studying gene function in eukaryotes by conditional gene inactivation. Annu Rev Genet. 36:153-73.

Structure of the Group

Group Leader Technical Assistants Dr. Manfred Gossen Dr. Maren Mieth Marion Papst Scientists Dr. Mathias Hampf* * part of the period reported

Graduate Students Tina Baldinger Pierre Debs Weimin Liu Anand Ranjan 86

Evolution, Regulation, and Genetic 2. We propose to exploit transposons to determine the iden- Applications of Transposable tity, function and biological relevance of genes that are Elements in Vertebrates associated with vertebrate embryonic development and human disease, by isolating their counterparts from model organisms such as fish, frogs, and mice. Specifically, we Zoltán Ivics are in the process of : a) introducing both directed and random mutations into the transposase gene in the hope that we can derive hyperactive versions of the transposon system. With such hyperactive vectors, we hope to be able to efficiently knock out genes in vertebrate model organisms; b) initiating an FP transposon-based insertional mutagenesis screen in the zebrafish, using gene-trap transposon vectors whose expression is dependent on transposition into transcribed genes. Spatial and temporal patterns of reporter expression can be co-localized with phenotypic changes in developing zebrafish embryos; c) conducting a transposon-based misexpression screen in mammalian cells in order to identify novel genes involved in tumorigenesis.

3. We are investigating the molecular interactions between Project Description transposons and host cells. a) We are following a “candidate” approach by looking for Work in the “Transposition” group involves transposable interactions with cellular factors that are involved in DNA elements. We follow two main areas of research: 1) other recombination systems. We established that high Molecular biology of DNA transposition in vertebrate cells; mobility group proteins, as well as proteins that are and 2) Applications of transposable elements in vertebrate involved in double-strand DNA break repair, are host genetics. In the past years, we have laid the foundation for factors of transposition. We are in the process of inve- using Sleeping Beauty (SB) and Frog Prince (FP) as mole- stigating the involvement of other repair and/or cell cular tools to address both of these areas. cycle checkpoint proteins in transposition. b) We also follow a “blind” approach by performing a In search for host-encoded factors that participate in, or regu- yeast two-hybrid screen on a human gene library. With late, the transposition reaction, we identified the DNA-bend- the screen, we have already identified two human prote- ing protein HMGB1 as a cofactor of SB transposition. ins that specifically interact with the Sleeping Beauty HMGB1 most likely plays a role by assisting synaptic com- transposase. plex formation during transposition. Furthermore, we have c) In search of gene regulatory networks that are activated shown that proteins involved in double-strand break repair in response to transposition, we are in the process of are limiting factors of transposition in mammalian cells and identifying relevant transcriptional changes of gene that both homology-dependent and homology-independent expression by using Affymetrix gene chips. This repair pathways contribute to the success of a transposition approach allows us to gain insight into the complex event. On the front of vector development for insertional regulation of transposition in vertebrate cells. mutagenesis and gene therapy, we developed improved SB- based vectors that show an almost 10-fold increase in transpositional efficiency when compared to the first-generation Selected Publications vectors. We have shown unprecedented gene-trapping efficiencies with FP transposition in mammalian cells, Izsvák, Zs. and Ivics, Z. (2004). Sleeping Beauty transposi- thereby demonstrating the usefulness of this vector system for tion: Biology and applications for molecular therapy. Mol. functional gene analyses in vertebrate species. Ther. 9:147-156.

Izsvák, Zs., Stüwe, E.E., Fiedler, D., Katzer, A., Jeggo, P.A. We are currently concentrating our efforts on the following and Ivics, Z. (2004). Healing the wounds inflicted by projects: Sleeping Beauty transposition by double-strand break repair in mammalian somatic cells. Mol. Cell 13:279-290. 1. SB has a number of advantages as a gene vector when compared to current viral and non-viral gene transfer Zayed, H., Izsvák, Zs., Khare, D., Heinemann, U. and Ivics, technologies. Our goal is to evaluate, develop, and modify Z. (2003). The DNA-bending protein HMGB1 is a cellular the SB vector system so that it will become an efficient and cofactor of Sleeping Beauty transposition. Nucleic Acids Res. safe vector for human gene therapy. Specifically, we are in 31:2313-2322. the process of determining the rate at which transposon vectors integrate into chromosomes of non-dividing cells. 87

The Sleeping Beauty transposable element and its transposition. (A) Components of the element. On top, a schematic drawing of the transposon is shown. The element has a single gene encoding the transposase, which is flanked by terminal inverted repeats (IR/DRs, blue arrows), each containing two binding sites for the transposase (small green arrows). A sequence alignment shows the actual sequences of the external and internal transposase binding sites. The transposase has an N-terminal, bipartite, paired-like DNA-binding domain containing a GRRR AT-hook motif, a nuclear localization signal (NLS) and the DDE catalytic domain. (B) Transposition. The transposase gene within the element can be replaced by a therapeutic gene, and the resultant transposon can be maintained in a simple plasmid vector. The transposase is supplied in trans. The transposase binds to its binding sites within the IR/DR repeats and, together with host factors such as HMGB1, forms a synaptic complex, in which the ends of the transposon are paired. The transposon is excised from the donor molecule and integrates into a new location.

Miskey, Cs., Izsvák, Zs., Plasterk, R.H. and Ivics, Z. (2003). The Frog Prince: a reconstructed transposon from Rana pipiens with high activity in vertebrates. Nucleic Acids Res. 31:6873-6881.

Izsvák, Zs., Khare, D., Behlke, J., Heinemann, U., Plasterk, R.H. and Ivics, Z. (2002). Involvement of a bifunctional, paired-like DNA-binding domain and a transpositional enhancer in Sleeping Beauty transposition. J. Biol. Chem. 277:34581-34588.

Vigdal, T., Kaufman, C., Izsvak, Z., Voytas, D. and Ivics, Z. (2002). Common physical properties of DNA affecting target site selection of Sleeping Beauty and other Tc1/mariner transposable elements. J. Mol. Biol. 323:441452.

Structure of the Group

Group Leader Dr. Zoltán Ivics

Scientists Dr. Zsuzsanna Izsvák Dr. Chris Kaufman

Graduate and Undergraduate Students Technical Assistants Hatem Zayed Andrea Katzer Oliver Walisko Siegne Knespel Csaba Miskey Mareike Becker Secretariat Baghat Christ Verona Kuhle

Guest Scientist Akira Hikosaka Structural and Functional Genomics

Structural Studies of Proteins have determined the crystal structure of domain I of PI-SceI at and Nucleic Acids high resolution in order to clarify some open questions about the protein’s DNA-binding mode (Werner et al., 2002). The by X-ray Crystallography structure suggested a sub-domain motion in domain I to be required for site-specific DNA binding. This model was Udo Heinemann qualitatively supported subsequently by other research groups.

The process of partitioning ensures that dividing bacterial cells receive a full complement of the chromosomal or plasmid DNA. Low-copy-number plasmids often encode a Biological processes in normal and diseased cells are best un- ParA/ParB pair of proteins guiding this process, where ParA derstood utilizing three-dimensional structures of the large is an ATPase and ParB is a site-specific DNA-binding protein. molecules upon which they are based. Macromolecular In collaboration with E. Lanka (MPI for Molecular Genetics, crystallography is a uniquely powerful tool to study the struc- Berlin), we are studying the structural basis of partitioning of tures of proteins, nucleic acids and their complexes, since it the conjugative Escherichia coli plasmid RP4 whose proteins permits the determination of the precise arrangement of all IncC and KorB serve as ParA and ParB homologs. As a first atoms and the shape and property of all surfaces in single step towards a full structural characterization of the system, molecules as well as huge molecular complexes. This know- the crystal structure of the C-terminal domain of KorB ledge can be used to explain biochemical observations, to pre- (KorB-C) was determined (Delbrück et al., 2002). KorB-C dict biological functions, and to design ligands specific to a was shown to be a closely linked dimer with subunits of SH3- site on the surface of given protein molecule. We combine like fold, and solution and mutagenesis data indicate that the X-ray diffraction studies with biochemical and biophysical C-terminal domain is indeed responsible for the dimerization studies of proteins involved in nucleic-acid binding or linked of intact KorB. More recently, the structure of the central part to disease states, in particular in the cancer field. Crucial to of KorB, KorB-O, bound to a 17-bp DNA fragment contain- this work is the ability to prepare crystallizable samples of ing the consensus KorB-binding sequence (OB, present 12 nucleic acids, proteins, and protein domains by chemical, bio- times on RP4) was determined by X-ray diffraction methods. chemical, and gene-technological means. The underlying KorB-O was shown to be a completely -helical protein that techniques have recently been adapted to medium-to-high binds the DNA through major-groove contacts. Surprisingly, throughput in the frame of the Protein Structure Factory, the the specificity-determining contacts to the binding site are first European structural genomics initiative. formed by amino-acid residues outside the helix-turn-helix motif of KorB. These findings were corroborated by mutagenesis studies. Nucleic acid-interacting proteins

The homing endonuclease PI-SceI is an intein, an internal Structural genomics of human proteins protein, embedded into the extein sequence of the vacuolar membrane H+-ATPase. In a protein splicing process, it relea- Structural genomics is defined as a world-wide “large-scale ses itself from the precursor and combines the two exteins to project to determine the three-dimensional shapes of all a functional ATPase. Responsible for this activity is domain I proteins and other important biomolecules encoded by the ge- of PI-SceI, which also contributes most of the energy required nomes of key organisms”. The Max Delbrück Center is par- for specific binding to a DNA sequence of at least 31 bp ticipating in this new approach to structural biology by length, the VMA1-vde locus on chromosome 8 of Saccha- assuming a leading role in the Berlin-area Protein Structure romyces cerevisiae. This locus is the allele of a VMA1 gene Factory (Heinemann et al., 2003). This consortium focuses on (VMA: vacuolar membrane H+-ATPase) that is deficient of the systematic structure analysis of human proteins and deve- vde (VDE: VMA-derived endonuclease = PI-SceI). In a pro- lops high-throughput technology for expression cloning, process called “homing”, domain II of PI-SceI cuts a specific tein crystallization, and synchrotron-based X-ray diffraction recognition sequence at the VMA1-vde locus and initiates experiments. We have used the Protein Structure Factory the insertion of the vde gene. PI-SceI thus has dual activities approach to study a number of human proteins that are related in catalyzing both protein and DNA splicing reactions. In to cancer and/or play important roles in cellular signaling or collaboration with A. Pingoud (University of Gießen), we transport processes. 89

Structures of human proteins determined within the Protein Structure Factory. Clockwise from top left: Crystal structures of nicotinamide mo- nonucleotide adenylyltransferase (NMNAT), gankyrin, a fumarylacetoacetate-family hydrolase member, hp14.5, the PX domain from p47phox, Bet3p, and prolyl peptidase D. Center: NMR structure of the C-terminal BRCT domain from BRCA1 (left) and crystal structure of -spectrin SH3 domain (right).

The human protein hp14.5 is a member of the large (Manjasetty et al., 2004b). A sequence motif, LXCXE YjgF/YER057c/UK114 protein family which comprises more (178LACDE182 in gankyrin), known to interact with Rb is in than 200 members. The gene encoding hp14.5 is down-regu- -helical conformation, whereas it was found in extended, lated in kidney and liver tumors, whereas a high expression -like structure in two different Rb-bound proteins. The level is observed in fully differentiated cells. L-PSP, the rat crystal structure of gankyrin may serve as a starting point homolog of hp14.5, is known to inhibit protein synthesis in towards a full structural and biochemical study of gankyrin- vitro, probably due to its endoribonucleolytic activity towards ligand interactions. single-stranded RNA. The crystal structure shows hp14.5 to be a symmetric trimer composed of chorismate-mutase-like In vesicle transport between cellular compartments, docking subunits (Manjasetty et al., 2004a). Surface clefts at the sub- or tethering complexes residing on the target membrane me- unit interfaces are lined with amino-acid residues conserved diate early steps of vesicle attachment that precede the pairing in the YjgF/YER057c/UK114 protein family. These clefts of SNARE proteins and membrane fusion. The TRAPP bind benzoate molecules from the crystallization buffer and (transport protein particle) docking complex is involved in are likely to function as active sites carrying a hydrolytic tethering of ER-derived COP-II vesicles to the cis-Golgi com- activity of the protein. partment. It consists of at least seven different polypeptides and can be immunoprecipitated with one labeled subunit, Gankyrin is a recently described oncoprotein linked to hepa- Bet3p. The crystal structure of Bet3p shows this protein to be tocellular carcinoma. The protein is primarily composed of dimeric with subunits of -plait topology. Each subunit is ankyrin repeats and interacts with a number of molecules, covalently modified with a palmitate molecule covalently some of which are known to play important roles in cell coupled to the sulfhydryl group of Cys68 via a thioester signaling and cancer. Among these are the retinoblastoma linkage. The palmityl moieties are deeply buried inside the protein (Rb), the cyclin-dependent protein kinases CDK4 and protein, thus keeping it soluble in aqueous buffers. The struc- CDK6, the melanoma antigen (MAGE-A4), and the S6 AT- ture suggests, however, a mechanism by which the fatty acid Pase of the 26S proteasome. Gankyrin counter-acts the kinase chains may be extruded from Bet3p into the Golgi membrane inhibitor p16INKA4, also an ankyrin-repeat protein, and the- bilayer. It appears possible that the palmitoylation of Bet3p is reby modulates the CDK4-mediated Rb phosphorylation, involved in fixing the TRAPP complex to the target mem- reducing the stability of Rb. In the crystal, gankyrin is present brane. The Bet3p structure opens the exciting possibility to as a monomeric protein with an extended surface formed by structurally characterize a complete tethering complex or sub- five complete and two terminal, incomplete ankyrin repeats complexes thereof. 90

Selected Publications

Manjasetty, B.A., Delbrück, H., Pham, D.-T., Mueller, U., Fieber-Erdmann, M., Scheich, C., Sievert, V., Büssow, K., Niesen, F., Weihofen, W., Loll, B., Saenger, W. & Heinemann, U. (2004a) Crystal structure of Homo sapiens protein hp14.5. Proteins: Struct. Bioinf. 54, 797-800.

Manjasetty, B.A., Quedenau, C., Sievert, V., Büssow, K., Niesen, F., Delbrück, H. & Heinemann, U. (2004b) X-ray structure of human gankyrin, the product of a gene linked to hepatocellular carcinoma. Proteins: Struct. Funct. Bioinf. 55, 214-217.

Heinemann, U., Büssow, K., Mueller, U. & Umbach, P. (2003) Facilities and methods for the high-throughput crystal structure analysis of human proteins. Acc. Chem. Res. 36, 157-163.

Werner, E., Wende, W., Pingoud, A. & Heinemann, U. (2002) High resolution crystal structure of domain I of the Saccha- romyces cerevisiae homing endonuclease PI-SceI. Nucleic Acids Res. 30, 3962-3971.

Delbrück, H., Ziegelin, G., Lanka, E. & Heinemann, U. (2002) An Src homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promis- cuous IncP plasmid RP4. J. Biol. Chem. 277, 4191-4198.

Structure of the Group

Group Leader Prof. Dr. Udo Heinemann

Scientists Dr. Katja Faelber* Dr. Michael Kolbe Dr. Babu A. Manjasetty Dr. Jürgen J. Müller Dr. Andrew P. Turnbull

Graduate and Undergraduate Students Technical Assistants Sarbani Bhattacharya Anette Feske Ralf Bienert Andreas Knespel Kerstin Böhm Gisela Sklenar* Heinrich Delbrück* Olaf Gaiser Administrative Assistant Andrei Halavaty Birgit Cloos Dheeraj Khare Ravi Kumar Lokareddy Klaas Max Yvette Roske Gisela Tünnemann* Erik Werner* 91

Computer Simulation of for selective recognition of specific base sequences by regu- Biomolecular Structures, latory proteins. Structural libraries, derived from the analysis Dynamics, and Interactions of experimentally solved structures and modeling results, allow for a structural description of binding sites for specific transcription factors and help in the search for sites with Heinz Sklenar characteristic and common features in long sequences with unknown function.

We have participated in an international initiative with the goal to improve the underlying data by using large-scale Molecular Dynamics simulations on the current state-of-the- art level. For this purpose, 39 DNA fragments, each 15 base pairs long and including all possible tetranucleotides, have been selected and simulated over 15 ns. Preliminary results, however, show that the simulation time is still too short for full conformational equilibration. It was therefore suggested that the simulations be repeated using our new Monte Carlo (MC) technique. Performance and results of this approach were demonstrated by applications to three DNA dodecamers

with the palindromic sequences d(CG)6, d(TA)6, and d(CGC- GAATTCGCG). In case of palindromic sequences, the degree of equilibration is indicated by the differences observed for Computer simulations are based on physical models that equivalent base pair steps. Compared with sequence-induced describe the driving forces for the formation of molecular effects, such differences are already very small after 106 MC structures. The results lead to a better understanding of bio- cycles, which need less than one week of CPU time on a molecular structures in terms of their physical properties, help currently available PC. Fast equilibration of counterions was to predict what structures are formed, and how these struc- found to be important for observing frequent conformational tures interact in living systems. The computational approach transitions in the DNA oligomers. The averaged structures complements high-resolution structure determination using show the characteristics of B-form DNA with sequence- X-ray crystallography and NMR spectroscopy. dependent helical step parameters that are close to the averages calculated for the ten different dinucleotide steps from In the last years, we have focussed on the development of a crystallographic databases. new simulation technique based on the Monte Carlo Metro- polis algorithm. The advantage of this approach, in comparison with conventional Molecular Dynamics, was shown in DNA-ligand interactions applications to sequence-dependent DNA structures and their Remo Rohs interactions with ligands and suggests that it too can be successfully used for simulations of protein structures and Methylene blue, an efficient singlet oxygen generating dye, biological membranes. binds to DNA and allows photosensitized reactions to be used for sequence-specific cleavage of the DNA backbone. Inter- calation and groove binding are possible binding modes of the Monte Carlo simulation algorithm dye, depending on base sequences and environmental condi- Daniel Wüstner

By using the constant bond lengths approximation and solv- ing the chain breakage/closure problem in the bond/torsion Currently implemented molecular model for Monte Carlo simulations of nucleic acid structures, including 14 independent chain and ring variables per nucleotide. The P-O5’ angle space, collective variables have been defined that main- and O1’-C4’ bonds are chosen for chain breakage/closure, where the positions of tain structural moves entirely locally and allow for large atoms O5’ and C4’ are determined by the closure equations. conformational changes in Monte Carlo simulations. It is essentially this choice of independent variables that permits conformational equilibration on a reasonable computational time scale, which is a necessary condition for deriving meaningful structural data from the trajectories of molecular simulations. The algorithm has been implemented in several new programs for the simulation of nucleic acid structures, ligand-DNA complexes, and biological model membranes.

Sequence dependent structure and dynamics of DNA

Subtle sequence effects on the helical geometry and dynamics of DNA structures have been found to be critically important 92

tions. According to experimental results, the dye intercalates sidered as plant-specific ZFPs. Most of the unique aZFPs are into GpC and CpG base pair steps, but prefers minor groove derived from extensive duplication events in the Arabidopsis binding in case of AT alternating DNA sequences. We have genome, resulting in several expanded ZFP families. The two therefore extended our former modeling study of methyl-ene largest families, named C1 and A1, are constituted by 64 and blue binding to a DNA decamer with AT alternating base se- 24 members, respectively, and are suggested to be involved in quence, in order to compare both sequences. The results show transcriptional regulation. that our modeling technique faithfully reproduces the experimental data and, moreover, has enabled us to explain the sig- The largest lineage specific ZFP families in Drosophila and nificantly different binding behavior in energetic and structu- Arabidopsis described above are completely unrelated and ral terms. Although the relative stability of the different reflect the diversity of transcriptional regulation guided by complexes is similar for the two sequences, subtle differences ZFPs in animals and plants. In contrast, the few conserved are seen in energy decompositions and can be attributed to the ZFPs, found in animals and plants, should be involved in change from symmetric 5’-YpR-3’ intercalation to minor more ancient molecular processes like RNA metabolism and groove binding with increasing salt concentration, which is chromatin remodeling. experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. This difference is due to a significantly lower non-electrostatic energy for the Selected Publications minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a Rohs, R. and Sklenar, H. (2004) Methylene Blue binding to higher deformation energy of DNA (including solvent contri- DNA with alternating AT base sequence: Minor groove bind- butions). The energetic data are in agreement with the conclu- ing is favored over intercalation. J. Biomol. Struct. Dyn. 21, sions derived from different spectroscopic studies and can 699-711. also be structurally interpreted on the basis of the modeled complexes. Sklenar, H. and Rohs, R. (2003) A new chain breakage/ closure algorithm yields fast conformational equilibration of nucleic acid structures in Monte Carlo simulations. J. Biomol. Conservation, diversification, and expansion of Struct. Dyn. 20, 835-836. C2H2 zinc finger proteins in eukaryotic genomes Siegfried Böhm Chung, H.R., Schäfer, U., Jäckle, H., and Böhm, S. (2002) Genomic expansion and clustering of ZAD- containing C2H2 C2H2 zinc finger proteins (ZFPs) constitute the most abun- zinc-finger genes in Drosophila. EMBO Rep. 3, 1158-1162. dant family of nucleic acid binding proteins in eukaryotes. The basic functions of C2H2 ZFPs range from DNA or RNA Rohs, R. and Sklenar, H. (2001) Methylene Blue binding to binding to their involvement in protein-protein interactions. DNA with alternating GC base sequence: Continuum treat- In addition, a comparison of the whole ZFP sets in the main ment of salt effects. Ind. J. Biochem. Biophys. 38, 1-6. eukaryotic lineages has revealed a further level of ZFP complexity through their remarkable lineage specific diversification and expansion. Structure of the Group

In the period reported, we have analyzed the full ZFP sets in Group Leader the Drosophila and Arabidopsis genomes (dZFPs and aZFPs), Dr. Heinz Sklenar with the aim of their classification in functional terms.This work was done in close collaboration with the groups of H. Scientists Jäckle (MPI Göttingen) and W. Mewes (IFB München). In the Dr. Siegfried Boehm Drosophila genome we have identified 326 putative ZFP Dr. Daniel Wüstner* genes corresponding to ~2.3% of all annotated genes. 94 of the dZFPs are conserved in other animals. In addition, another Graduate Students ~1/3 of unique dZFPs contain a novel N-terminal zinc-finger- Remo Rohs* associated domain (ZAD), which is restricted to insects. ZAD-ZFPs constitute the most expanded subfamily of dZFPs Technical Assistant and are clustered at few chromosomal sites.These features are Werner Leistner reminiscent of the vertebrate specific KRAB-ZFPs. Based on the very recently solved crystal structure of the ZAD domain, * part of the period reported it was shown that ZAD domains are involved in protein- protein interactions like the KRAB domains, suggesting their possible repressor function.

In the Arabidopsis genome we have identified 175 putative ZFP genes corresponding to ~0.7% of all annotated genes. Only 31 of the aZFPs are conserved in other eukaryotes. Their vast majority (144 out of 175) is unique to Arabidopsis. They are largely conserved in other plants and can therefore be con- 93

Nucleosides, Nucleotides, and liferating tissues (Fig. 1). This and a second promising [18F]- Oligonucleotides PET precursor will be pursued with the aim to get an approval for clinical application.

Eckart Matthes Inhibitors of HBV and HCV replication

Recently, L-nucleosides, the stereoisomeric analogs of the naturally occurring D-nucleosides have gained tremendous interest as highly selective inhibitors of viral infections. In line with this development, we have focussed our interest on hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. In cooperation with H. Will (Heinrich-Pette Institute, Hamburg) and R. Bartenschlager (Molecular Virology, Otto- Meyerhof Center, University Heidelberg), we have designed and synthesized new L-deoxyribonucleoside and L-ribonucleosides targeting HBV DNA polymerase and RNA- dependent RNA polymerase (NS5B), respectively. Some highly selective inhibitors of HBV replication in HepG2 2.2.15 cells were detected and presented in a patent application.

To date, no L-ribonucleosides are known with antiviral A new nucleoside-marker for tumor imaging by activity against HCV. Surprisingly, we identified two compo- PET unds which inhibit HCV-replication in a Huh-7 cell-based replicon assay. They display only minimal effects on cellular [18F]-Fluorodeoxyglucose ([18F]FDG) is currently the most RNA synthesis and cellular proliferation. Thus, further widely used radiotracer for imaging malignant tumors by positron emission tomography (PET). However, its application in cancer diagnosis, staging, and therapy monitoring is limited by the fact that it is more an indicator of glucose Figure. Whole-body PET scan taken 1 h after i.v. application of 20 MBq [18F]-FAT. The coronal slice demonstrates the [18F]-FAT accumulation in the tumor located in the left metabolism than of tumor proliferation.The development of flank (arrow). more specific cancer imaging agents is currently the focus of intensive research.

In a common project with the Clinic for Nuclear Medicine, Charité – University Medicine Berlin (D. L. Munz), we have designed, synthesized, and investigated a series of modified cranial thymidine analogs which meet biological and chemical requirements we consider essential for successful tumor imaging. Principally, such nucleoside analogs should be resistant against the nucleoside degrading enzyme thymidine phos- phorylase and should be phosphorylated well by thymidine kinase 1, an enzyme whose activity is increased 10-20 fold in proliferating cells. The product of this enzyme reaction is a nucleoside monophosphate which is trapped inside of proliferating cells and can be detected by PET provided that the applied thymidine analog is labeled appropriately, e.g. with lr [18F].

We have found one promising new nucleoside which meets these requirements (FAT) and for which we have established a rapid and simple synthesis and a carrier-free nucleophilic [18F]- labeling and purification procedure. All these methods were adapted successfully to an automated PET tracer synthe- sizer (Nuclear Interface).

These results enable us to apply the new PET precursor to the caudal detection of tumors in mice. Whole-body PET scans taken from mice with a subcutaneously grown malignant melanoma (B16) 1 h after i. v. application of 20 MBq [18F]-FAT have shown a highly significant uptake of the [18F] activity in the tumor compared with non-proliferating as well as with pro- 94

chemical modifications are directed toward increasing their Structure of the Group antiviral activity. Group Leader Dr. Eckart Matthes Telomerase detection by an optical biosensor Scientists We have found earlier that phosphorothioate modified Dr. Martin von Janta-Lipinski primers bind strongly to the primer binding site, a specific Dr. Dieter Bärwolff protein motif of telomerase resulting in a dramatic increase of the velocity of the enzyme reaction in comparison to unmodi- Graduate and Undergraduate Students fied primers. Yong Wu* Peter Lahnert* In cooperation with the Fraunhofer Institute for Biomedical Lora Dimitrova Techniques, Potsdam (F. Bier), we put these findings to prac- tical use for the development of a new fiberoptical biosenso- Technical Assistants ric system to detect telomerase activity in human tumors in Klaus Gaertner real time without PCR. Christine Lehmann* Inge Krahn For this purpose, FITC-labeled probes which were comple- Helga Scheer* mentary to the newly synthesized telomeric DNA were added. Jürgen Schildt* For detecting signals, an evanescent-wave assay configu- Marianne Stulich* ration was selected to take advantage of the total internal reflection fluorescence. Unpurified extracts of 106 HL-60 * part of the period reported tumor cells were found to be sufficient for a selective telomerase proof. Thus, our aim is to make this method more sensitive.

Selected Publications

Schmidt, P.M., Lehmann, C., Matthes, E., and Bier, F. F. (2002). Detection of activity of telomerase in tumor cells using fiber optical biosensors. Biosens. Bioelectron. 17, 1081-1087.

Schmidt, P.M., Matthes, E., Scheller, F. W., Bienert, M., Lehmann, C., Ehrlich, A., and Bier, F. F. (2002). Real-time determination of telomerase activity in cell extracts using an optical biosensor. Biol. Chem. 383, 1659-1666.

Harnack, U., Lehmann, C., Matthes, E., and Pecher, G. (2001). Upregulation of telomerase activity in Herpesvirus saimiri immortalized human T-lymphocytes. Anticancer Res. 21, 3969-3972.

Schmidt, P.M., Matthes, E., Scheller, F. W., and Bier, F. F. (2001). Nachweis der Telomeraseaktivität in Zellkulturen mittels eines faseroptischen Sensors. Sensorik 47-51.

Mentel, R., Kurek, S., Wegner, U., von Janta-Lipinski, M., Gurtler, L., and Matthes, E. (2000). Inhibition of adenovirus DNA polymerase by modified nucleoside triphosphate analogs correlate with their antiviral effects on cellular level. Med. Microbiol. Immunol. 189, 91-95. 95

Tumor Genetics Comprehensive analysis of German breast/ovarian cancer families for BRCA1/BRCA2 germline mutations: Mutation profiles and frequencies in the Siegfried Scherneck German population W. Hofmann, E. Claßen, D. Horn, B. Jandrig, S. Seitz, A. Nothnagel, I. Sümnich, H. Zeidler, K. Krause, A. Pietsch- mann, M. Hinzmann, the “Berlin Center for Hereditary Breast and Ovarian Cancer” in cooperation with the “German Consortium for Hereditary Breast and Ovarian Cancer (GCHBOC)”

Germline mutations in the BC susceptibility genes, BRCA1/ BRCA2, jointly explain the most significant part of the familial breast/ovarian cancer syndrome. Within the GCHBOC, which was initiated and supported by the “Deutsche Krebs- hilfe”, we have participated in a comprehensive study to analyze the entire coding sequence of the BRCA1 and BRCA2 genes in about 3,000 patients from German breast/ovarian cancer families. A total of about 100 BRCA1 and about 80 BRCA2 distinct deleterious mutations have been identified by the GCHBOC. More than one third of these mutations are novel and might be specific for the German population. The The research program of this group aims to better understand mutation study has defined groups of high-risk families. the genetic basis of cancer, particularly human breast cancers Mutation rates of only 50% and lower in the higher-risk (BC). BC, one of the most common cancers affecting women, groups provide evidence for further predisposing genes. At has been demonstrated to be a complex genetic disease with present, identified mutations and unclassified variants in the high clinical heterogeneity and characterized by the accumu- BRCA genes, as well as specific rearrangements in mutation lation of multiple molecular alterations. At present, our know- carriers, have been characterized for clarification of geno- ledge about BC associated genes, their functions, and inter- type-phenotype correlations. In addition, the prevalence and actions in pathways regulating growth and arrest of BC cells penetrance of mutations of other genes for hereditary breast is still limited. cancer, like CHEK2, have been analyzed.

Although about 90% of BC are sporadic, the remaining cases are heritable and caused by mutations of at least two tumor Systematic search for genes contributing to the suppressor genes, BRCA1 and BRCA2. It is likely that more genesis and progression of sporadic and hereditary highly penetrant susceptibility genes will emerge. However, breast cancer attention is now shifting toward more common low-pene- S. Seitz, B. Jandrig, M. Plaumann, A. Schwartz, C. Zeller, K. trance mutations and their possible contribution to BC. There- Wenzel, P. Waßmuth, D. Siele, J. Strissel, R. Frege, K. fore, multiple approaches, such as linkage in high-risk fami- Poppe, S. Werner, in cooperation with A. Meindl (München), lies and association studies in large BC case-control studies, N. Arnold (Kiel), D. Niederacher (Düsseldorf), K. Schmutzler are being used to identify additional high- and low-pene- (Köln), P.M. Schlag, (RRK, Berlin), I. Petersen (Berlin), B. trance genes. Hinzmann, A. Rosenthal (metaGen, Berlin), W. Arnold (atugen, Berlin). Furthermore, new technological developments offer powerful means to analyze the activity of thousands of genes in sam- Over the last few years, we have focused our research on ples of sporadic and hereditary BC at the DNA, RNA, and genes whose function is impaired or lost during BC develop- protein levels. Such analyses have proven to be valuable in ment on chromosome regions 6q24 (i.e. SASH1), 8p12-p21 identifying genes and pathways associated with different (i.e. TRAIL-receptor genes, EXTL3) and 17p13.3 (i.e. types of BC, response to treatment, and prognosis. PFN1). Some of the somatic genetic alterations (genes) identified have been correlated with clinico-pathological parameters.

To identify and validate BC associated genes, several positional and functional approaches are being used in combination: identification of differentially expressed ESTs by electronic and real Northern blotting and RT-PCR; fine mapping of LOH hotspots; microarray-technology; and mutation analysis. In addition, transfection assays and functional complementation tests have been applied. 96

Recently, we have developed a promising strategy to search Structure of the Group for BC relevant genes by combining array based expression profiling of BC with a powerful functional approach; namely, Group Leader the microcell mediated chromosome transfer in BC cells. We Prof. Dr. Siegfried Scherneck identified a common set of candidate genes which can be placed in different pathways. Several of the genes and path- Scientists ways identified may prove to be useful in diagnosis and pro- Elvira Claßen* vide new targets for therapies directed against a BC tumor Dr. Wera Hofmann type. Dr. Denise Horn* Dr. Burkhard Jandrig Tumor suppressor activity of the microfilament pro- Dr. Irina Lapidous tein profilin 1 in breast cancer Dr. Marlies Plaumann B. Jandrig, I. Lapidous, A. Schwartz, K. Rücker, A. Rejman Dr. Arnfried Schwartz Lipinski, M. Blankenburg, in cooperation with B.M. Dr. Susanne Seitz Jockusch, M. Rothkegel (Braunschweig), W. Arnold (atugen, Dr. Katrin Wenzel* Berlin), P.M. Schlag (RRK, Berlin) Graduate and Undergraduate Students Profilin 1 (PFN1) is a regulator of the microfilament system Michaela Blankenburg* and involved in various signaling pathways. Immunohisto- Maria Hinzmann* chemical analysis revealed intermediate and low levels of Anette Rejman-Lipinski* profilin 1 in different human BCs. Dagmar Siele* Jana Strissel* The importance of PFN1 for human tissue differentiation has Peter Waßmuth* been demonstrated by our research findings that human BC Constanze Zeller* cells expressing low PFN1 levels adopt a non-tumorigenic phenotype upon raising their profilin-1 level. In a study to Technical Assistants characterize the ligand binding sites crucial for PFN1 tumor Renate Frege suppressor activity in BC, we found that the actin binding site Kerstin Krause* is instrumental for this activity. Konstanze Poppe Karin Rücker Ingeborg Sümnich* Selected Publications Sabine Werner Helga Zeidler Zeller, C., Hinzmann, B., Seitz, S., Prokoph, H., Burkhard- Goettges, E., Fischer, J., Jandrig, B., Estevéz-Schwarz, L., Guest Scientists Rosenthal, A., and Scherneck, S. (2003) SASH1: a candidate Dr. Mehmet Gögülü tumor suppressor gene on chromosome 6q24.3 is downregu- Dr. Dr. Konrad Kölble lated in breast cancer. Oncogene 22, 2972-2983. Andrea Pietschmann

German Consortium for Hereditary Breast and Ovarian Can- Secretariat cer (Hofmann, W., Horn, D., and Scherneck, S. for the Center Renate Galle of Berlin) (2002) Comprehensive analysis of 989 patients with breast or ovarian cancer provides BRCA1 and BRCA2 * part of the time reported mutation profiles and frequencies for the German population. Int. J. Cancer 97, 472-480.

Thompson, D., Easton, D.F., Seitz, S., Scherneck, S., and the Breast Cancer Linkage Consortium (2002) Cancer incidence in BRCA1 mutation carriers. J. Natl. Cancer Inst. 94, 1358- 1365.

Seitz, S., Wassmuth, P., Fischer, J., Nothnagel, A., Jandrig, B., Schlag, P.M., and Scherneck, S. (2002) Mutation analysis and mRNA expression of TRAIL-receptors in human breast cancer. Int. J. Cancer 102, 117-128.

Janke, J., Schlüter, K. Jandrig, B., Theile, M., Kölble, K., Arnold, W. Grinstein, E., Schwartz, A., Estevéz-Schwarz, L., Schlag, P.M. Jockusch, B., and Scherneck, S. (2000) Suppres- sion of tumorigenicity in breast cancer cells by microfilament protein profilin 1. J. Exp. Med. 191, 1675-1685. Tumor Immunology

Differentiation and Growth Control the B cell areas of lymphoid organs. Chemokines are also in Lymphocyte Development and responsible for fine-tuning the localization of lymphocytes within microcompartments. B cell positioning at the bound- Immunopathogenesis ary of B cell follicle and T cell zone in the spleen, where naïve, mature B cells interact with T cells recently activated Martin Lipp in the adjacent T cell zone, has been shown to be controlled by the balanced responsiveness of B cells towards the ligands for CCR7 and CXCR5.

In addition to their function in the organization of lymphoid organ microarchitecture, CXCR5 and CCR7 are crucial for Chemokines are essential regulators of lymphocyte migration lymphoid organogenesis. Mice deficient for the chemokine throughout the body. The chemokine system controls lympho- receptor CXCR5 or its ligand CXCL13 lack several types of cyte recirculation in immune system homeostasis as well as peripheral lymph nodes and the majority of Peyer’s patches. the activation-dependent and tissue-selective trafficking of In addition, both strains of mice show severe alterations in the lymphocytes and dendritic cells during immune responses. In morphology of the spleen and the remaining Peyer’s patches. addition, chemokines are critical factors for the development In contrast, secondary lymphoid organ development is largely and organization of secondary lymphoid organs. Our main unaffected in mice lacking the expression of the chemokine focus is the role of homeostatic chemokine receptors in lym- receptor CCR7 or in mice lacking the expression of its ligands phoid organ development, systemic immune responses, and within secondary lymphoid organs. However, by generating chronic inflammatory diseases. In addition, we are interested mice with a targeted deletion for both chemokine receptors, in the immune modulatory and growth-inducing functions of we have shown that CCR7 cooperates with CXCR5 in lym- chemokine receptors encoded by human herpesviruses, and phoid organ development. the function of sphingophospholipid receptors in the immune system. The expression of CCR7 and CXCR5 defines functionally distinct T cell subsets Development and organization of lymphoid tissues by homeostatic chemokine receptors CCR7 is the dominant chemokine receptor mediating T cell entry and positioning within secondary lymphoid organs. Its Lymphocyte homing to lymphoid and nonlymphoid tissues, expression is tightly regulated during T cell differentiation as well as lymphocyte recirculation between secondary lym- from naïve towards memory/effector cells: Expression of phoid organs, critically depends on the chemokine system. CCR7 and CD62L, which are necessary for homing to secon- According to their expression pattern and function in immune dary lymphoid tissues and highly expressed on naïve CD4+ T system homeostasis and immune responses, chemokines are cells, are downregulated on terminally differentiated effector broadly divided into two groups - homeostatic and inflamma- cells. Instead, effector cells express chemokine receptors for tory chemokines. Although there exists some redundancy in inflammatory chemokines that allow them to enter nonlym- the chemokine system, chemokine receptor expression and phoid tissues at sites of infection and inflammation. Within receptor sensitivity on lymphocytes, as well ligand expres- peripheral blood, expression of CCR7, CXCR5, and CD62L sion in lymphoid and peripheral tissues, appears to be tightly allow for the identification three functionally distinct subsets regulated. Expression of the chemokine receptors CCR7 and of memory/effector CD4+T cells. CCR7–CD62L– effector

CXCR5 enables T cells, B cells, and dendritic cells to enter memory T (TEM) cells have downregulated CCR7 and most secondary lymphoid organs. In addition, they are responsible closely resemble classical effector cells that preferentially for the proper positioning of these cells within distinct home to nonlymphoid organs. In contrast, expression levels microenvironments of lymphoid organs. For example, CCL19 of CCR7 and CD62L remain high on a second subset of me- and CCL21, ligands for CCR7, which are expressed by den- mory/effector cells. These cells appear to retain the capacity dritic cells and stromal cells within the T cell zones of secon- to home to secondary lymphoid organs and have therefore dary lymphoid organs, retain T cells within this microenvi- been named central memory T (TCM) cells. A third population, ronment. In contrast, the ligand for CXCR5, CXCL13, is about 15% of human peripheral blood CD4+ T cells, expres- expressed by follicular dendritic cells and stromal cells within ses CXCR5 along with CCR7 and was provisionally named

B cell follicles and attracts B cells and subsets of T cells into TCM1. Within secondary lymphoid organs, the population of 98

CXCR5+CD4+ T cells is significantly enlarged. The upregula- in the development of transplant rejection. Remarkably, in a tion of CXCR5 is accompanied by a downregulation of CCR7 model of acute allogeneic tumor rejection, CCR7-/- mice com- and, consequently, these cells are able to enter B cell follicles. pletely failed to reject subcutaneously injected MHC class I CD4+CXCR5+ T cells located within germinal centers ex- mismatched tumor cells and cytotoxic activity of allospecific press costimulatory molecules such as ICOS and CD154 and T cells was severely compromised. When solid tumors act as B helper T cells in that they promote the antibody secre- derived from wild type mice were transplanted, recipient tion by B cells. For this reason, we have named these cells fol- CCR7-/- mice were capable of rejecting the allografts. In con- -/- licular T helper B (TFH) cells. However, the origin and fate of trast, tumor allografts transplanted from CCR7 donors onto CXCR5+CD4+ T cells is still under discussion. Currently, we CCR7-/- recipients showed allograft survival up to 28 days, are analyzing these CD4+ memory/effector T cell populations suggesting a critical function of CCR7 on donor-type passen- in order to better understand their differentiation pathway and ger leukocytes in the initiation of cytotoxic CD8+ T cell their role in chronic inflammatory and infectious diseases. responses. In a heterotopic heart transplantation model, CCR7 deficiency resulted in significantly prolonged, but not indefinite, allograft survival. Additional prolongation of graft CCR7 controls cytotoxic T cell priming in survival was observed when hearts from CCR7-/- mice were alloimmune responses used as donor organs. Our results define a key role for CCR7 (in cooperation with J. Droese; H.-G. Zerwes, Novartis, in allogeneic T cell priming within the context of draining Basel) lymph nodes.

The requirement of CCR7, which regulates co-localization of T cells and mature dendritic cells within secondary lymphoid Immune modulatory and growth-inducing functions organs, in efficient priming of allo-specific cytotoxic CD8+ T- of viral chemokine receptors cells is poorly characterized. We could demonstrate a critical role for CCR7 in the initiation of an alloimmune response and We have previously shown that Epstein-Barr-Virus (EBV) specifically transactivates expression of the cellular chemokine receptor CCR7 by its regulatory nuclear factor EBNA2. In contrast to EBV, several other human herpesviruses, like Figure 1 Model for the role of CXCR5 and CCR7 during early steps of lymph node and cytomegalovirus (CMV) or the lymphotropic human herpes- Peyer’s patch development. The accumulation of RORt+ IL-7R+ lymphoid tissue virus type 6 (HHV-6) and Kaposi’s sarcoma-associated her- inducer (LTi) cells, which derive from a population of fetal liver cells, and mesenchymal pesvirus (KSHV), also termed HHV-8, encode viral chemo- organizer (MC) cells is probably driven by a positive feedback loop. IL-7R+ LTi cells stimulated via IL-7R or TRANCE-R upregulate surface expression of LT 1 2. The kine receptors and chemokines in their genomes suggesting stimulation of mesenchymal organizer cells in the developing lymph node or Peyer’s that herpesviruses use the chemokine system to interfere with patch anlage by IL-7R+ LTi cells via LTR induces the expression of adhesion molecules, such as VCAM-1, ICAM-1, MadCAM-1 and chemokines, including CXCL13, the growth and differentiation program of the host and sub- CCL19, and most probably CCL21. In turn, CXCL13 and CCL21 leads to the activation vert specific immune responses. of 4 1 or 4 7 integrin on LTi cells, which reinforces the interaction of inducer and organizer cells and facilitates continuous signaling through LTR. The accumulation of LTi cells and their tight interaction with mesenchymal organizer cells eventually reaches a critical size or level of chemokine expression that allows lymph node and Peyer’s patch development to proceed to the next stage which includes the immigration of A murine model for Kaposi´s sarcoma other cell types such as B cells, T cells, and macrophages, as well as their organization (in cooperation with E. Kremmer, GSF; I. Anagnostopoulos, into distinct microenvironments (Nat. Immunol., 5, 12-14, 2004). H. Stein, FU; K. Kölble, Charité)

Infection with HHV-8 has been linked by epidemiological and molecular evidence to the pathogenesis of all forms of Kapo- si’s sarcoma (KS), a non-Hodgkin’s B cell lymphoma, and multicentric Castleman’s disease (MCD). The research project aims to establish whether the HHV-8-encoded chemokine receptor (vGPCR) plays a critical role in the development of HHV8-associated diseases and malignancies as an essential oncogenic and paracrine factor. Using a newly developed vGPCR-specific monoclonal antibody, significant expression of the viral chemokine receptor could be confirmed in all virus-associated human diseases.

The HHV-8-encoded vGPCR has been implicated in the pathogenesis of Kaposi’s sarcoma particularly because of its high constitutive signaling activity. We have used retroviral transduction to generate vGPCR-expressing NIH3T3 cell lines that are tumorigenic in nude mice but, as expected, fail to induce tumors in their immunocompetent counterparts. However, tumor fragments obtained from nude mice grow progressively in immunocompentent BALB/c mice. Unex- pectedly, vGPCR expressing cells established from grafted tumor fragments give rise to angioproliferative fibrosarcomas 99

Figure 2 Altered primary B cell follicles in the spleen of CXCR5–/–, CCR7–/– and CXCR5–/–CCR7–/– mice. Cryostat sections were stained with PE-labeled anti-CD3 (red), FITC-labeled anti-IgD (green) and biotinylated anti-IgM/Streptavidin-Cy5 (blue) to visualize the architecture of primary B cell follicles. In wild-type mice, marginal zone B cells (IgMhiIgDlo) appear as a rim around the polarized clusters of follicular B cells, which are mainly IgMloIgDhi. In CXCR5–/– mice, B cells fail to organize in polarized clusters. The T cell zone is instead surrounded by a ring of IgM-IgDhi B cells, which is in turn encompassed by a thickened ring of IgMhiIgDlo marginal zone B cells. CCR7–/– mice generally lack prominent T cell zones in the white pulp. In contrast, T cell zones and B cell zones are largely disorganized in CXCR5–/–CCR7–/– mice. White arrows indicate the position of the IgMhiIgDlo marginal zone B cells; WT, wild type; T, T cell zone; B, B cell zone; Original magnification: x200 (Immunol. Rev. 195, 117-135, 2003)

in immunocompetent mice, which exhibit a striking histolo- Function and signaling of Cytomegalovirus gical resemblance to KS including spindle cell morphology, a (CMV)-encoded chemokine receptor US28 high degree of vascularization and brisk mitotic activity. High (in cooperation with A. Rehm, J. Droese and B. Dörken) expression of the vGPCR was confirmed in both the cell lines and tumors by vGPCR-specific staining. This novel animal The HCMV encoded receptor US28 may play an important model of KS might contribute to the understanding of the role in the pathogenesis of herpesvirus infections through underlying molecular mechanisms promoting vGPCR-asso- binding and sequestering of extracellular inflammatory ciated oncogenesis and immune evasion and will facilitate the -chemokines. This project is aimed at elucidating the US28 development of vGPCR-specific vaccination strategies. signaling pathways underlying chemotaxis and chemokinesis in CMV infected cells. US28 displays constitutive activation of both phospholipase C and NF-kappaB signaling and exhibits a high basal level of phosphorylation independently from 100

ligand binding. We elucidated that such unique constitutive Mokros, T., Rehm, A., Droese, J., Oppermann, M., Lipp, M., receptor phosphorylation is a prerequisite for the subcellular and Höpken, U.E. (2002). Surface expression and endocytosis localization and for the rapid agonist-independent endocyto- of the human cytomegalovirus-encoded chemokine receptor sis of the receptor. In contrast to all other chemokine recep- US28 is regulated by agonist-independent phosphorylation. J. tors, US28 was found to internalize in a clathrin-coated pit Biol. Chem. 277, 45122-45128. dependent, but arrestin-independent, manner. Currently, we are investigating the protective antiapoptotic pathways that Reif, K., Ekland, E.H., Ohl, L., Nakano, H., Lipp, M., Förster, may be employed by US28 expression to maintain survival of R., and Cyster, J.G. (2002). Balanced responsiveness to virus infected cells. chemoattractants from adjacent zones determines B-cell position. Nature 416, 94-99.

Role of sphingophospholipid receptors in the immune system Structure of the Group

EDG receptors represent a novel family of G protein-coupled Group Leader receptors, binding either lysophosphatidic acid (LPA) or Dr. Martin Lipp sphingosine 1-phosphate (S1P). Five receptors, S1P1 (EDG- 1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6), and S1P5 Scientists (EDG-8), have been identified as high affinity receptors for Dr. Felix Cifire S1P in mammals. S1P exerts a variety of responses, including Dr. Linda Diehl* proliferation, differentiation, and migration, by activating a Dr. Uta E. Höpken distinct set of G proteins coupled to the S1P receptors in Dr. Gerd Müller different types of cell. Our laboratory has identified and Dr. Christian Ried characterized S1P4, which is expressed specifically in cells Dr. Tobias Schulze and tissues of the hematopoietic and lymphoid system. Recently, we have analyzed signaling pathways mediated via Graduate and Undergraduate Students S1P4 and have shown that S1P4 couples directly to G i and Jemina Benga very potently to G 12/13-subunits of trimeric G-proteins. Con- Sebastian Geibel* sequently, S1P4 induces pertussis toxin-sensitive PLC activa- Sven Golfier tion and Rho-GTPase-dependent cytoskeletal rearrangements Frank Jeblonski* such as peripheral stress fiber formation and cell rounding Azita Mahiny upon S1P stimulation. The capacity of S1P receptors to Thilo Mokros mediate fundamental responses such as cell motility and Ata-Ur Rasheed shape change through G i- and G 12/13-coupled signaling Philipp Reiterer pathways suggests an important in vivo role for the S1P Isaac Sipo* system in the control of cell migration in the context of the Nanthakumar Thirunarayanan tissue microenvironment in lymphocyte homeostasis and Antje Wengner acute and chronic inflammatory immune responses. In order Frank Wilde -/- to explore the in vivo function of S1P receptors, S1P4 mice have been generated are currently under investigation. Technical Assistants Dagmar Breitfeld Jenny Gorsch Selected Publications Peter Graßhoff* (Operator) Kerstin Krüger Lipp, M., and Müller, G. (2004) Lymphoid organogenesis: Dagmar Meyer* getting the green light from RORt. Nat. Immunol., 5, 12-14 Heike Schwede Dr. Peter Rahn (Operator) Höpken, U. E., Droese, J., Li, J.-P., Joergensen, J., Breitfeld, D., Zerwes, H.-G., and Lipp, M. (2004) The chemokine Secretariat receptor CCR7 controls lymph node-dependent cytotoxic T Daniela Keyner cell priming in alloimmune responses. Eur. J. Immunol., 34, 461-470 * part of the period reported.

Prinz, M., Heikenwalder, M., Junt, T., Schwarz, P., Glatzel, M., Heppner, F.L., Fu, Y.X., Lipp, M., and Aguzzi, A. (2003). Positioning of follicular dendritic cells within the spleen controls prion neuroinvasion. Nature 425, 957-962.

Gräler, M.H., Grosse, R., Kusch, A., Kremmer, E., Guder- mann, T., and Lipp, M. (2003). The sphingosine 1-phosphate receptor S1P4 regulates cell shape and motility via coupling to Gi and G12/13 . J. Cell. Biochem. 89, 507-519. 101

Biology of Hodgkin’s Lymphoma Characterization of disease-associated genes in Hodgkin’s lymphoma Bernd Dörken Martin Janz, Stephan Mathas in cooperation with Rudolf Manz (Deutsches Rheumaforschungszentrum), Christian Ha- gemeier (Charité) and Harald Stein (Charité)

Malignant transformation of hematopoietic cells is associated with profound alterations in the transcriptional program resulting in deregulated proliferation, differentiation, and apoptosis. Using classical Hodgkin’s lymphoma (cHL) as a model system, we are investigating the role of transcription factors in the oncogenic process. Using oligonucleotide micorarrays, we have generated expression profiles for cHL- derived cell lines as well as Non-Hodgkin B-cell lines. In addition, we have analyzed the expression pattern of primary non-malignant human B- and T-cell populations. Our microarray data provide the basis for the identification of differentially expressed genes that determine the malignant phenotype of cHL. Candidate genes are further validated by in situ hybridization and immunohistochemistry on primary tumor tissue. This experimental strategy revealed that ATF3, a member of the CREB/ATF family of transcription factors that is Scope involved in the cellular response to stress signals and has been shown to negatively regulate p53 function, is strongly over- Hodgkin and Reed-Sternberg cells are tumor cells of classical expressed in primary Hodgkin cells. Using cell lines that Hodgkin’s lymphoma. In most cases they are derived from constitutively overexpress ATF3, as well as vector-based germinal center B cells. However, they do not express immu- siRNA constructs that downregulate ATF3 expression, we are noglobulins and typical B-cell markers on their cell surface. investigating the significance of ATF3 in malignant growth We focus our work on the characterization of the molecular and survival of lymphatic cells. basis for the dedifferentiated B-cell phenotype of Hodgkin’s lymphoma and try to identify molecular defects that might be responsible for tumor cell transformation. Biology of Hodgkin’s lymphoma and multiple myeloma Franziska Jundt, Kristina Schulze-Pröbsting, Katharina Kley Role of c-FLIP and AP-1 in the pathogenesis of in cooperation with Harald Stein (Charité) Hodgkin’s lymphoma Stephan Mathas, Andreas Lietz, Franziska Jundt, Martin Notch1 belongs to a family of transmembrane receptors that Janz in cooperation with Claus Scheidereit (MDC) and Harald control cell proliferation and differentiation in response to Stein (Charité) extracellular ligands expressed on neighbouring cells. The Notch1 gene has been described as being involved in a trans- As a molecular defect responsible for proliferation and location in a human acute T-cell lymphoblastic leukemia/lym- apoptosis resistance of Hodgkin-/Reed-Sternberg (HRS) phoma, and, its constitutively active form, produces T-cell cells, we identified a constitutive activity of transcription fac- neoplasms in mice. However, a pathogenetic role for Notch1 tor NF-B. In an attempt to establish inhibition of the NF-B in B-cell neoplasms was so far unknown. We showed that activity as a treatment option for Hodgkin’s lymphoma (HL), Notch1 is strongly expressed in B-cell derived HRS cells and we were able to show a strong anti-tumor activity of arsenic in tumor cells of T-cell derived anaplastic large cell lympho- containing compounds in vitro and in vivo in a mouse model. ma (ALCL). Our data indicate that activation of Notch1 Furthermore, we could demonstrate that NF-B dependent signaling in tumor cells by its ligand Jagged1 regulates tumor up-regulation of c-FLIP proteins is the key mechanism for de- cell growth and survival and suggest that pharmacological ath receptor resistance of HRS cells, including inhibition of manipulation of the Notch1 system might have therapeutic the CD95 and TRAIL receptor pathways. Ongoing work aims potential in these lymphomas. to identify the mechanisms leading to constitutive NF-B activity in HRS cells and to develop clinically applicable Furthermore, Notch receptors are expressed on hematopoietic treatment options for HL based on NF-B inhibition. A stem cells and interact with their ligands on bone marrow further project of the group is the analysis of the recently stromal cells and, thereby, control cell fate decisions and sur- described constitutive AP-1 activity in HRS cells. In this vival. We investigated whether Notch signaling is involved in project, AP-1 activation mechanisms and target genes are the tight interactions between neoplastic plasma cells and investigated. This also includes the analysis of these tran- their bone marrow microenvironment, that are essential for scription factors with respect to the dedifferentiation of HRS tumor cell growth in multiple myeloma (MM). We demon- cells. strated that Notch receptors and their ligand Jagged1 are highly expressed in cultured and primary MM cells, whereas non- neoplastic counterparts show low to undetectable levels of 102

High Notch1, Notch2 and Jagged1 expression in primary MM cells. (A-H) Bone marrow biopsy specimen of one case of MM. (A) Giemsa staining demonstrating bone marrow infiltration by atypical plasma cells (MM cells) with large nuclei and prominent nucleoli. (B) MM cells show monotypic expression of immunoglobulin light chain kappa. Immunostains for myeloperoxidase (C) and glycophorin C (D) mark granulocytic and erythroid precursors showing that MM cells are surrounded by non-malignant bone marrow cells. (E, F) Double labeling for CD138 (brown reaction product; streptavidin/biotin/peroxidase technique) and Notch1 (red reaction product; immunoalkaline-phosphatase method) reveals that CD138 positive MM cells strongly co-express Notch1. (G, H) Double labeling for CD138 (brown reaction product) and the Notch ligand Jagged1 (red reaction product) demonstrates coexpression of CD138 and Jagged1 on MM cells. (F, H) Some CD138 negative non-malignant bone marrow cells, mainly megacaryocytes, show weak immunoreactivity against Notch1 or Jagged1. (I-K) Extramedullar (liver) biopsy specimen of MM immunostained for Notch1 (I), Notch2 (J) and Jagged1 (K) and counterstained with hematoxylin. MM cells are intensely labeled by anti-Notch1, anti-Notch2 and anti-Jagged1 antibodies. (L-N). Isolated plasma cells (CD38+++/CD19+) of non-neoplastic bone marrow of normal donors (cytospins) show low to undetectable immunoreactivity against Notch1 (L), Notch2 (M) and Jagged1 (N). Original magnification x 200.

Notch. Functional data indicate that ligand-induced Notch the apoptotic Mcm3 fragments, suggesting a self-enforcing signaling is a growth factor for MM cells and suggest that mechanism by which proteolyzed caspase targets could con- these interactions contribute to lymphomagenesis of MM in tribute to the perpetuation of the apoptoticsignaling cascade. vivo. We are now establishing the DT40 cell line model to test the function of the Mcm3 and Cdc6 fragments in a conditional knock out system. Identification of molecular regulators involved in B cell apoptosis Barbara Tiedt, Ute Nitschke, Kurt Bommert in cooperation Selected Publications with Manfred Gossen (MDC) Jundt, F., Schulze-Pröbsting, K. Anagnostopoulos, I., Apoptotic downregulation of DNA replication is one of the Muehlinghaus, G., Chatterjee, M., Mathas, S., Bargou, R.C., possible mechanisms to ensure the fast and accurate execu- Manz, R., Stein, H. and Dörken, B. (2004). Jagged1-induced tion of cell death. It might support the acceleration of DNA Notch signaling drives proliferation of multiple myeloma fragmentation and the saving of energy needed for the cells. Blood, Jan 15 [Epub ahead of print]. apoptotic processes. DNA replication itself is a highly organized and regulated process, which is initiated by binding of the Mathas, S., Lietz, A., Janz, M., Hinz, M., Jundt, F., Scheide- pre-replicative complex (pre-RC) to the origins of DNA repli- reit, C., Bommert, K., and Dörken, B. (2003) Inhibition of cation. Cdc6 and the MCM proteins (MCM2-7) are integral NF-B is essential for arsenic induced apoptosis. Blood parts of the pre-RC. For two of the components, Mcm3 and 102:1028-1034. Cdc6, we could show an apoptotic cleavage. The cleaving enzymes and the exact cleavage sites utilized in response to Jundt, F., Anagnostopoulos, I., Förster, R., Mathas, S., Stein, diverse apoptotic stimuli are analyzed for both proteins. The H., and Dörken, B. (2002) Activated Notch1 signaling pro- intracellular localization and chromatin association of wild- motes tumor cell proliferation and survival in Hodgkin and type Mcm3 and Cdc6 in comparison to caspase-resistant mu- anaplastic large cell lymphoma. Blood 99:3398-3403. tants and to the apoptotic protein fragments are analyzed. Furthermore, we could show a pro-apoptotic effect for one of 103

Jundt, F., Kley, K., Anagnostopoulos, I., Schulze Pröbsting, K., Greiner, A., Mathas, S., Scheidereit, C., Wirth, T., Stein, H., and Dörken, B. (2002) Loss of PU.1 expression is associated with defective immunoglobulin expression in Hodgkin and Reed-Sternberg cells of classical Hodgkin disease. Blood 99:3060-3062.

Mathas, S., Hinz, M., Anagnostopoulos, I., Krappmann, D., Lietz, A., Jundt, F., Bommert, K., Mechta-Grigoriou, F., Stein, H., Dörken, B., and Scheidereit, C. (2002) Aberrantly expressed c-Jun and JunB are a hallmark of Hodgkin lymphoma cells, stimulate proliferation and synergize with NF-B. EMBO J. 21:4104-4113.

Structure of the Group

Group Leader Prof. Dr. Bernd Dörken

Scientists Dr. Kurt Bommert Dr. Florian Emmerich* Dr. Martin Janz Dr. Franziska Jundt Dr. Stephan Mathas

Graduate and Undergraduate Students Andreas Lietz Kristina Schulze-Pröbsting Barbara Schories Sabine Friedl

Technical Assistants Franziska Hummel Pia Herrmann Katharina Kley Kerstin Krüger Brigitte Wollert-Wulf Ute Nitschke

* part of the period reported 104

Mechanisms of Immune Evasion in Role of the HCMV encoded chemokine receptor Tumor Biology and in Herpes Virus US28 in immune evasion Infections (in cooperation with U.E. Höpken, T. Mokros*, N. Lehmann, M. Lipp)

Armin Rehm (Helmholtz Fellow) The HCMV encoded chemokine receptor, US28, serves as a decoy for inflammatory chemokines. We could show that rapid capture and endocytosis of chemokines relies on an agonist-independent phosphorylation at the intracellular C-terminus of the receptor. Receptor phosphorylation controls the rate of surface deposition, which is usually very low. In contrast to all other chemokine receptors, HCMV encoded US28 was found to internalize in a clathrin-coated pit dependent, but arrestin-independent manner. Furthermore, we could elucidate the structural motifs necessary for direct coupling of the receptor to the clathrin-coated pit associated adaptor molecules. Currently, we are exploring protective antiapoptotic pathways that may be employed in US28 expressing cells.

Physiological and pathophysiological role of the tumor-associated antigen RCAS1/ EBAG9 Scope (in cooperation with U.E. Höpken and C. Birchmeier-Kohler)

A major focus of our work is related to the role of immuno- Truncated O-linked glycan epitopes, among Tn and TF surveillance in tumor biology and Herpes virus infections. (Thomsen-Friedenreich), are a hallmark of essentially all Viruses have evolved several mechanisms to employ their types of experimental and human cancers. They are thought to hosts’ metabolic pathways to evade immune attack, also be associated with cell adhesion, invasion, and metastasis of referred to as “stealth function”. A second area of interest tumor cells. includes the mechanisms tumor cells employ to elude the immune system. We apply cell biological methods, as well as We have identified a ubiquitously expressed Golgi-localized animal models, to study the pathophysiological role of a molecule, RCAS1/EBAG9, which modulates Tn and TF sur- recently identified tumor-associated antigen shown to alter face expression and was found to be expressed in high abun- glycan structures at the cell surface of tumor cells. dance in secretory active tumor tissues. The identification of an interaction partner points to a more physiological role in the secretory pathway, since the SNARE-associated molecule Viral interference with the MHC class I antigen Snapin was identified. In vivo, we study the consequences of presentation pathway a genetic deletion of RCAS1/EBAG9 in a mouse knock out model where we focus on the generation of glycan epitopes, Immunosurveillance is mediated by an innate immune but also on the exocytosis function in endocrine and neuronal response followed by the adaptive arm of the immune system cells. Correspondingly, the modulatory role of RCAS1/ for greater efficiency. Human cytomegalovirus encodes genes EBAG9 in vitro in the exocytotic pathway is under scrutiny. which inhibit MHC class I mediated antigen presentation. Among them, the US11 and US2 gene products dislocate efficiently MHC class I heavy chains out of the ER and into Association of lymphoma dissemination and the cytosol where they are degraded by the proteasome. We chemokine receptor expression have been able to show that the membrane glycoprotein US11 (in cooperation with U.E. Höpken, M. Lipp, and I. Anagnosto- exhibits an unusual, delayed posttranslational signal peptide poulos, UKBF) cleavage, which is presumably correlated with its immuno- evasive function. US2 and US11 are not redundant but exhibit The underlying mechanisms of migration and invasion of a significantly different efficiency in class I degradation when tumor cells often recapitulate those that are effective in non- probed in human dendritic cells. We have generated transge- neoplastic cells during physiological processes, among them nic mice that express their transferred genes in a tissue-speci- immune-cell trafficking. It has been established that lym- fic, tetracycline inducible manner. This model will allow us to phocyte trafficking is largely orchestrated by the interaction assess the proposed in vivo function of US11. of chemokine receptors and their cognate ligands. In this context, primary-mediastinal B-cell lymphoma are generally considered as a subgroup of diffuse large B cell lymphoma- however, they exhibit completely different patterns of dissemination and recurrence. Since they share a number of surface markers compared to thymic B cells, a close relationship was assumed. In this project, we take advantage of the chemokine/chemokine receptor system to assess functionally and 105

phenotypically the development of thymic B cells and we will correlate the profiles obtained from PMBL cell lines and tissues with their supposed ancestors. This approach will help to elucidate novel molecular markers for PMBL, but it might also help to define a pathogenetic trait of PMBL development.

Selected Publications

Engelsberg, A., Hermosilla, R., Karsten, U., Schülein, R., Dörken, B., and A. Rehm (2003). The Golgi protein RCAS1 controls cell surface expression of tumor-associated O-linked glycan antigens. J. Biol. Chem. 278: 22998-23007

Rehm, A., Engelsberg, A., Tortorella, D., Körner, I.J., Leh- mann, I., Ploegh, H.L., and U.E. Höpken (2002). Human cytomegalovirus gene products US2 and US11 differ in their ability to attack major histocompatibility class I heavy chains in dendritic cells. J. Virol. 76: 5043-5050

Mokros, T., Rehm, A., Droese, J., Oppermann, M., Lipp, M., and U.E. Höpken (2002). Surface expression and endocytosis of the human cytomegalovirus-encoded chemokine receptor US28 is regulated by agonist-independent phosphorylation. J. Biol. Chem., 277: 45122-45128

Rehm, A., P. Stern, H.L. Ploegh, and D. Tortorella (2001). Signal peptide cleavage of a type I membrane protein, HCMV US11, is dependent on its membrane anchor. EMBO J., 20: 1573-1582

Structure of the Group

Group Leader Dr. Armin Rehm

Scientists Dr. Tatiana Reimer Dr. Arne Engelsberg*

Graduate Students Constantin Rüder Jana Droese

Technical Assistant Insa Lehmann

* part of the period reported 106

Identification of Target Structures Based on these studies, we plan to develop novel molecular for the Development of therapeutic strategies. Molecular Therapies in Malignant B-cell Disorders Cytotoxic T-cell targeting by bispecific antibodies Michael Grün, Anja Löffler, Ralf Bargou in cooperation with Ralf C. Bargou Gerd Riethmüller (München) and Patrick Baeuerle (Micromet GmbH, München)

We have shown that a novel recombinant bispecific single chain antibody, bscCD19xCD3, induces rapid and high lymphoma directed cytotoxicity mediated by unstimulated T-lymphocytes. By redirecting primary human T cells derived from the peripheral blood against CD19-positive lymphoma cell lines, the bscCD19xCD3 antibody showed significant cytotoxic activity at very low concentrations even in experiments without T cell prestimulation. In addition, the bscCD19xCD3 bispecific antibody is able to induce nearly complete depletion of primary lymphoma cells mediated by autologous T-cell of patients with chronic lymphatic leukemia in the majority of cases analyzed. Furthermore, we could demonstrate that this novel antibody construct is also effec- Scope tive in animal models. Based on these preclinical data, we have conducted a phase-I clinical trial for the treatment of The aim of our research is to identify target structures for the patients with refractory B-cell lymphomas which was development of molecular therapies for malignant B-cell dis- finished at the end of 2003. Another study (phase I/II) will orders. Therefore, we pursue two different strategies. The first start in 2004. Furthermore, we are currently establishing a strategy focuses on functional characterization of growth re- similar strategy for the treatment of multiple myeloma gulating signaling pathways in multiple myeloma in order to patients using a novel plasmacell-specific surface antigen as a identify molecular targets for therapeutic intervention with target structure. small compound inhibitors. Another strategy focuses on the development of immunotherapies with recombinant antibody constructs for the treatment of B-cell Non-Hodgkin-lympho- Selected Publications mas. Based on our preclinical research, we are conducting experimental phase I/II clinical trials. Hönemann, D, Rimpler, M, Kufer, P, Chatterjee, M, Friedl, S, Riechert, F, Bommert, K, Dörken, B, and Bargou, RC (2004) A novel recombinant bispecific single-chain antibody, Signaling and survival pathways in multiple bscWue-1xCD3, induces T-cell mediated cytotoxicity to- myeloma wards human multiple myeloma cells. Leukemia, in press. Dirk Hönemann, Manik Chatterjee, Suzanne Lentzsch, Ralf Bargou in cooperation with Reinhold Schäfer and Christine Lentzsch, S, Gries, M, Bargou, R, Dörken, B, and Mapara, Sers (Institut of Pathology, Charité, Berlin) MY (2003) Macrophage inflammatory protein 1-alpha (MIP- 1a) triggers migration and signaling cacades mediating survi- The bone marrow microenvironment produces a number of val and proliferation in multiple myeloma cells. Blood 101: different survival factors that are important for the malignant 3568-3573. growth and drug resistance of multiple myeloma cells. One of the main factors reported to be important for survival and Löffler, A, Grün, M, Wuchter, C, Schriever, F, Kufer, P, growth of myeloma cells is IL-6. However, very recently we Dreier, T, Hanakam, F, Baeuerle, PA, Bommert, K, Karawa- could show that myeloma cells become independent of the jew, L, Dörken, B, and Bargou, RC (2003) Efficient elimina- IL-6-gp130-STAT-3 pathway if cells grow in the presence of tion of chronic lymphocytic lekaemia B-cells by autologous T their bone marrow microenvironment (BMM) suggesting that cells with a bispecific anti-CD19/anti-CD3 single-chain anti- the BMM stimulates additional IL-6-independent survival body construct. Leukemia 17: 900-909. pathways in multiple myeloma cells. Therefore, we are currently focusing on the identification and characterization Dreier, T, Baeuerle, P, Fichtner, I, Grün, M, Schlereth, B, of IL-6-independent pathways and IL-6-independent mecha- Lorenczewski, G, Kufer, P, Riethmüller, G, Gjörstrup, P, and nisms of cell growth and survival. We have experimental Bargou, RC (2003) T cell costimulus-independent and very evidence that Ras-triggered pathways, such as the MEK/ efficacious inhibition of tumor growth in mice bearing MAPK and the PI3K/AKT pathway, are activated in myeloma subcutaneous or leukemic human B cell lymphoma xeno- cells by IL-6 independent mechanisms and contribute to the grafts by a CD19-/CD3-bispecific single-chain antibody con- malignant phenotype of myeloma cells. Selective targeting of struct. J Immunol 170: 4397-4402. these pathways with either small compound inhibitors or siRNA constructs can induce cell death in myeloma cells. 107

Chatterjee, M, Hönemann, D, Lentzsch, S, Bommert, K, Sers, C, Herrmann, P, Mathas, S, Dörken, B, and Bargou, RC (2002) In the presence of bone marrow stromal cells human multiple myeloma cells become independent of the IL-6- gp130-STAT3 pathway. Blood 100: 3311-3318.

Structure of the Group

Group Leader Priv.-Doz. Dr. Ralf C. Bargou

Scientists Dr. Torsten Stühmer Dr. Suzanne Lentzsch*

Graduate Students Manik Chatterjee Dirk Hönemann Michael Grün

Technical Assistants Pia Hermann Margret Gries

* part of the period reported 108

Cell-biological Determinants of To investigate the key events of apoptosis at the mitochon- Treatment Response and Prognosis drial level in primary leukemia cells, we developed a flow- in Acute Leukemias cytometric method for simultaneous detection of mitochondrial cytochrome c release and caspase-3 activation, using a conformation-sensitive cytochrome c antibody and antibodies Wolf-Dieter Ludwig specifically detecting the cleaved fragment of caspase-3. The method proved to detect deficient mitochondrial apoptosis signaling in leukemic blasts from pediatric ALL patients, and identified the cytochrome c mediated caspase activation as the most predictive parameter for blast cell persistence on day 15 of induction therapy. These data indicate that constitutive differences in the activation of post-mitochondrial apoptosis signaling pathways significantly determine the initial response to treatment in childhood ALL (in cooperation with K.-M. Debatin, University of Ulm).

Identification of leukemia-associated immunophenotype (LAIP) of MRD cells and treatment response assessment in acute leukemia

Recent studies in acute leukemias have demonstrated that mo- Scope nitoring of MRD by flow-cytometric immunophenotyping and PCR techniques is useful for evaluating early response to Recent and ongoing research projects of the Acute Leukemia treatment and predicts clinical outcome. We, therefore, inve- Research Group have investigated cell-biological determi- stigated prospectively MRD detection by multi-parametric nants and mechanisms mediating drug resistance in leukemic flow cytometry and its impact in the disease monitoring in blasts as well as their prognostic impact in acute leukemias. ALL. A comprehensive panel of antigen markers has been These research projects have been embedded in the frame- established to reliably identify the LAIP of MRD cells. We work of internationally renowned multicenter trials in child- are now focusing on validation of this panel within the frame- hood (ALL-BFM) and adult (AMLCG) acute leukemias, the- work of ongoing European multicenter clinical trials in child- reby providing the opportunity to investigate biologically and hood ALL and on its application in the molecular characte- clinically well-characterized leukemic specimens, and to cor- rization of MRD cells by global gene expression analysis (see relate biologic findings with treatment response and progno- below). (in cooperation with: M. Dworzak, St. Anna Child- sis. We have especially focused on mechanisms of regulation/ ren’s Hospital Vienna, A. Biondi and G. Gaipa, University of deregulation of apoptosis, on detection of minimal residual Milano, G. Basso, University of Padua) disease (MRD), and on characterization of MRD cells using global gene expression profiling in acute leukemias. Gene expression analysis of leukemic blasts persisting during induction therapy Identification of apoptosis-related cell-biological determinants associated with therapy response Current studies on global gene expression in childhood ALL have analyzed a rather heterogeneous bulk of ALL blasts and Deregulated or deficient activation of apoptosis signaling do not enable the retrieval of specific information on treatment- pathways may account for treatment failure in acute leuke- induced gene expression changes in resistant or relapsed leukemias. Given the dramatically different clinical courses of pa- mic subclones, which may be “hidden” behind the bulk of tients with precursor T-cell acute lymphoblastic leukemia chemotherapy-sensitive leukemic blasts. Therefore, we have (ALL), we have been interested in apoptosis regulation in a chosen an alternative strategy aimed at direct investigation of large series of children with chemoresistant or chemosensi- gene expression in leukemic blasts persisting under prednisone tive disease and analyzed their susceptibility to cytotoxic on day 8 (i.e., prednisone poor response), a parameter with drug-induced apoptosis, cytokine responsiveness, and cyto- crucial prognostic significance in childhood ALL. To this end, kine modulation of apoptotic pathways induced by different an experimental approach has been established with sample drugs. Evaluation of these data in the context of immunophe- acquisition and identification of MRD blasts based on their leu- notype and clinical data revealed that precursor T-cell ALL kemia-associated immunophenotype as defined by multi-para- comprises several biologically distinct diseases and pointed metric flow cytometry. The approach has further included to a predictive value of in vitro apoptosis-related functional isolation of leukemic blasts by flow sorting, providing high- parameters for treatment response in vivo. In particular, our quality mRNAs. Most importantly, we have demonstrated that studies revealed maturation-dependent differences in the this experimental approach facilitates gene expression profiling regulatory pathways of apoptosis, suggesting that patients of patient samples with blast cell counts as low as 50-100 with a cortical thymocyte immunophenotype are much more cells/µl, thus even enabling investigation of patients with good responsive to drug-induced apoptosis as compared with response to prednisone. Intraindividual comparative analyses patients exhibiting an immature or mature immunophenotype. of matched “day 8” and “day 0” ALL samples revealed a set of 109

genes whose expression has been commonly changed in the “day 8” leukemic blasts. The observed changes have indicated a deregulated expression of certain signaling (e.g. STAT1, MAP3K8, CD13R)- and drug sensitivity (e.g. ABCA7)-related genes and suggested a preferential positioning of these cells in the early G1 cell cycle phase. These data illuminate the potential of this strategy to investigate treatment-specific gene expression changes and to identify molecular targets in treatment-resistant ALL blasts (in cooperation with C. Hagemeier, K. Seeger, Charité, Berlin CancerNet)

Selected Publications

Ratei R, Ludwig WD (2003) Flow-cytometry methods for the detection of residual leukemia. In: Leukemia and Lymphoma: Detection of Minimal Residual Disease, Zipf TF, Johnston DA, Editor, Humana Press Inc.: Totowa, NJ. 1-19.

Kern W, Kohlmann A, Wuchter C, Schnittger S, Schoch C, Mergenthaler S, Ratei R, Ludwig WD, Hiddemann W, Hafer- lach T (2003) Correlation of protein expression and gene expression in acute leukemia. Cytometry 55B: 29-36.

Wuchter C, Ruppert V, Schrappe M, Dörken B, Ludwig WD, Karawajew L (2002). In vitro susceptibility to dexamethasone- and doxorubicin-induced apoptotic cell death in context of maturation stage, responsiveness to interleukin 7, and early cytoreduction in vivo in childhood T-cell acute lymphoblastic leukemia. Blood 99: 4109-15.

Borkhardt A, Wuchter C, Viehmann S, Pils S, Teigler-Schlegel A, Stanulla M, Zimmermann M, Ludwig WD, Janka-Schaub G, Schrappe M, Harbott J (2002). Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases. Leukemia 16: 1685-90.

Structure of the Group

Group Leader Prof. Dr. Wolf-Dieter Ludwig

Scientists Dr. Richard Ratei Dr. Leonid Karawajew

Graduate Students Peter Rhein Stefan Richter Richard Schabath Doreen Oltersdorf

Technical Assistants Rosemarie Hoffmann Mathilde Martin Marianne Dunken Christina Witt Grit Czerwony* Sandra Vierich*

* part of the period reported 110

Role of Apoptosis and Consequently, we are currently testing to what extent indivi- Senescence in Tumor Development dual cancer-derived p53-missense mutants and defects in the and Treatment Responses p53 activating DNA damage signaling cascade may produce biased decisions towards distinct p53 controlled effector functions such as apoptosis or cellular senescence prior to and af- Clemens A. Schmitt ter DNA damaging therapies.

p53 and p16INK4a control cellular senescence as a drug-responsive program that impacts on the outcome of cancer therapy

As previously reported, p53 and INK4a/ARF mutations promote tumorigenesis and drug resistance, in part, by disabling apoptosis. We now show that primary murine lymphomas also respond to chemotherapy by engaging a senescence program controlled by p53 and p16INK4a. Hence, tumors with p53 or INK4a/ARF mutations – but not those lacking ARF alone – respond poorly to cyclophosphamide therapy in vivo. Moreo- ver, tumors harboring a Bcl2 mediated apoptotic block undergo drug-induced cytostasis involving the accumulation of p53, p16INK4a, and senescence markers, and typically acquire Scope p53 or INK4a mutations upon progression to a terminal stage. Finally, mice bearing tumors capable of drug-induced senes- Defective cellular growth control is a pivotal feature of the cence have a much better prognosis following chemotherapy malignant cell. Driven by mitogenic oncogenes, transformed than those harboring tumors with senescence defects. There- cells select for mutations that disable ultimate failsafe pro- fore, cellular senescence contributes to treatment outcome in grams such as apoptosis or cellular senescence. Importantly, vivo. DNA damaging anticancer agents may sensitize to programmed cellular responses that overlap with anti-oncogenic fail- In a subsequent study, we applied several genome wide ap- safe mechanisms. Hence, inactivation of apoptotic pathways, proaches to primary lymphomas arising with distinct INK4a/ for example, may not only facilitate tumor development, but ARF lesions to obtain additional genomic prognosticators of might simultaneously co-select for resistance to anticancer treatment outcome. Indeed, using spectral karyotyping therapy as well. Dissecting the pathways that control and exe- (SKY), comparative genomic hybridization (CGH), and cute apoptosis and senescence is expected to identify critical fluorescence in situ hybridization (FISH) we find recurrent regulators that may be targeted by novel therapies. genomic alterations that refine the predictive value of INK4a/ ARF lesions on drug responses in vivo. Moreover, we identified cytogenetic markers in untreated tumors that were indi- Dissecting p53 tumor suppressor functions in vivo cative of subsequent mutations during therapy. These data illustrate how genomic information from human tumors may We previously demonstrated that loss of p53 function accele- complement established prognostic markers and may im- rates tumor development and impairs sensitivity to anticancer prove anticancer treatment strategies. therapy in vitro and in vivo. Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 Selected Publications functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Schmitt, C.A. (2003) Senescence, apoptosis and therapy – Using a mouse model harboring primary, genetically-modi- cutting the lifelines of cancer. Nature Rev. Cancer, 3: 286-295 fied myc-driven B cell lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-nega- Lee, S., and C.A. Schmitt (2003) Chemotherapy response and tive caspase 9 by retroviral modification of hematopoietic resistance. Current Opin. Genet. Dev., 13: 90-96 stem cells confers – like p53 loss – a selective advantage, and completely alleviates pressure to inactivate p53 during lym- Schmitt, C.A., J.S. Fridman, M. Yang, S. Lee, E. Baranov, phomagenesis. Despite their p53-null-like aggressive pheno- R.M. Hoffman and S.W. Lowe (2002) A senescence program type, apoptosis-defective lymphomas that retain intact p53 controlled by p53 and p16INK4a contributes to the outcome genes do not display the checkpoint defects and gross of cancer therapy. Cell, 109: 335-346 aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis could be identified as the only p53 Schmitt, C.A., J.S. Fridman, M. Yang, E. Baranov, R.M. Hoff- function selected against during lymphoma development, man and S.W. Lowe (2002) Dissecting p53 tumor suppressor whereas defective cell-cycle checkpoints and aneuploidy are functions in vivo. Cancer Cell, 1: 289-298 mere byproducts of p53 loss. 111

Schmitt, C.A. and S.W. Lowe (2001) Bcl-2 mediates chemoresistance in matched pairs of primary Eµ-myc lymphomas in vivo. Blood Cells Mol. Dis., 27: 206-216

Structure of the Group

Group Leader Priv. Doz. Dr. Clemens A. Schmitt

Scientists Dr. Soyoung Lee Dr. Andrej Mirossay Dr. Maurice Reimann Dr. Pascal Kahlem Bianca Teichmann

Graduate Students Melanie Braig Katja Ziegenhorn Henry Däbritz

Technical Assistants Katja Lankisch Ines Schildhauer Claudia Kalla 112

Clinical and Molecular Oncology in resistance to anticancer therapy. In theory, the inactivation of p53 should also result in clinical resistance. Nevertheless, the clinical consequences of p53 disruption in human cancer Peter Daniel have been discussed controversially. While there is few doubt that p53 disruption can serve as a key step during tumorigenesis, there is so far only controversial evidence that p53 by itself is a key factor in the development of clinical resistance to anticancer therapies. To this end, we have investigated the consequences of genetic defects in genes acting as effectors or inducers of p53 that trigger apoptosis and cell cycle arrest programs upon genotoxic stress. There, in both in vitro and in clinical settings, we established that disruption of the intrinsic apoptosis pathway results in resistance to anticancer therapy. We also found that inactivation of single regulatory genes is often insufficient to explain resistance phenomena. In contrast, clinical resistance to anticancer therapy and poor prognosis often arise when cell cycle and cell death signaling is impaired by multiple defects, e.g. upon disruption of p53 and Bax or APAF-1 or Bax and Rb pathway components.

Regulation of cell death by pro-apoptotic Bcl-2 Cell death and cell cycle deregulation in cancer family members and resistance to anticancer therapy Apoptosis is mediated through at least three major pathways Virtually all medical anticancer therapies rely on the induc- that are regulated by (1) the death receptors, (2) the mito- tion of cell cycle arrest or cell death in the malignant cells. chondria, and (3) the endoplasmic reticulum (ER). In most Consequently, the analysis of such genetic events allows for cells, these pathways are controlled by the Bcl-2 family of the identification of patients at risk for an insufficient res- proteins that can be divided into antiapoptotic and proapopto- ponse to treatment with chemotherapeutic drugs or ionising tic members. Although the overall amino acid sequence ho- irradiation, and poor survival. Such analyses provide a ratio- mology between the family members is relatively low, they nal basis for a molecular understanding of the response to an- contain highly conserved domains, referred to as Bcl-2 homo- ticancer therapies and the clinical use of cancer therapeutics. logy domains (BH1 to BH4) that are essential for homo- and The aim of the group is, therefore, to define genetic defects in heterocomplex formation as well as for their cell death indu- cancer that result in aggressive disease, poor prognosis, and cing capacity. Structural and functional analyses revealed that resistance to clinical cancer therapy. To this end, we have the proapoptotic homologs can be subdivided into the Bax established an extensive genotyping program in solid tumors subfamily and the growing BH3-only subfamily. and leukemias. Recent pharmacogenomic data obtained from these screenings depict that defects in central regulatory BH3-only proteins link upstream signals from different cellu- genes, e.g. of the p53 pathway, do not result in global resi- lar or functional compartments to the mitochondrial apoptosis stance to therapy but may be overcome by adequate therapeu- pathway (Figure 1). Cleavage of Bid by caspases-8 or -3, e.g. tic modalities. Functional consequences of such cell death during death-receptor-induced apoptosis generates tBid that and cell cycle defects are analysed in vitro, often by the use of adenoviral gene transfer for complementation of disrupted genes. In addition, these systems are exploited to gain insights into novel aspects of cell cycle and cell death regulation and their intricate interactions. Figure 1 Function of BH3-only proteins as death sensors.

Understanding resistance to anticancer therapy

Anticancer therapies, i.e. chemotherapy and ionising irradiation, activate nuclear stress responses to induce cell cycle arrest and DNA repair. When repair fails, the same stress responses trigger cellular senescence or death and demise of the affected cell. The molecular basis of these events has been studied extensively during recent years and comprehensive models are now established for large parts of these signaling events. Studies utilizing both in vitro and in vivo models in genetically defined cell line models or knock-out mice clearly demonstrated that inactivation of p53 (or of genes acting in the same signaling cascade, i.e. the p53 pathway) may result 113

Selected Publications

Gillissen B, Essmann F, Graupner V, Stärck L, Radetzki S, Dörken B, Schulze-Osthoff K, Daniel PT (2003) Induction of cell death by the BH3-only Bcl-2 homolog Nbk/Bik is mediated by an entirely Bax-dependent mitochondrial pathway. EMBO J; 22:3580-90.

von Haefen C, Wieder T, Essmann F, Schulze-Osthoff K, Dörken B, Daniel PT (2003) Paclitaxel-induced apoptosis in BJAB cells proceeds via a death receptor-independent, caspase-3/caspase-8-driven mitochondrial amplification loop. Oncogene; 22: 2236-47.

Rau B, Sturm I, Lage H, Berger S, Schneider U, Hauptmann S, Wust P, Riess H, Schlag PM, Dörken B, Daniel PT (2003) Dynamic expression profile of p21WAF1/CIP1 and Ki-67 predicts survival in rectal carcinoma treated by preoperative radiochemotherapy. J Clin Oncol; 18:3391-3401.

Figure 2 Immunocytochemistry for Nbk localisation in DU145 prostate carcinoma. Red: Hemmati PG, Gillissen B, von Haefen C, Wendt J, Stärck L, Nbk (localizes to the ER), green: mitochondria. Güner D, Dörken B, Daniel PT (2002) Adenovirus-mediated overexpression of p14ARF induces p53 and Bax-independent apoptosis. Oncogene; 21: 3149-61.

Güner D, Sturm I, Hemmati P, Hermann S, Hauptmann S, Wurm R, Budach V, Dörken B, Lorenz M, Daniel PT (2002) Multigene analysis of Rb-pathway and apoptosis-control in esophageal squamous cell carcinoma identifies patients with good prognosis. Int J Cancer; 103: 445-454.

binds to Bax. This triggers a conformational switch in Bax that exposes the Bax C-terminus, induces redistribution of Structure of the Group Bax to the mitochondria, and insertion into the outer mitochondrial membrane. In contrast, Bak is localized constituti- Group Leader vely in the outer mitochondrial membrane. Upon activation, Priv. Doz. Dr. med. Peter Daniel both Bax and Bak oligomerize and form channels that release cytochrome c. Bim is released from the motor-dynein com- Scientists plex whereas Bmf is released from actin-myosin filaments Dr. Bernhard Gillissen during apoptosis. Puma, Noxa, Hrk, and Nbk (Bik) are indu- Dr. Dilek Güner ced by p53 and mediate cell death originating from the Dr. Philipp Hemmati nucleus, e.g. upon DNA damage. Phosphorylation of Nbk by Dr. Silke Radetzki a so far undefined kinase enhances Nbk function. Nbk locali- Dr. Christian Scholz zes to the ER (Figure 2) and activates Bax indirectly, through Dr. Isrid Sturm a so far undefined ER-initiated death pathway (Gillissen et. Dr. Berlinda Verdoodt al., EMBO J 2003; 22:3580-90). In contrast, phosphorylation Jana Wendt of Bad through the Akt/PKB kinase inactivates Bad. The latter disrupts the binding of Bad to Bcl-xL and results in Bax Graduate Students and Bak-dependent apoptosis. Gaby Forro Clarissa von Haefen The aim of our work is to gain structural and functional insi- Anne Hasenjäger ghts into how these subfamilies promote or inhibit cell death Antje König signals and how these properties may be utilized for develop- Antje Müller ment of apoptosis-promoting cancer therapies. Our studies Guillaume Normand therefore deal with questions such as how cell cycle stress re- Lilian Stärck sponses including anticancer therapies and oncogene deregulation feed into the mitochondrial death pathway. The focus is Technical Assistants on the mechanism of activation of pro-apoptotic multidomain Anja Richter Bcl-2 homologs and their interaction with BH3-only proteins Antje Richter that link various upstream signals including death receptors Jana Rossius and DNA damage signalling to the mitochondrial and the ER Sylvia Scheele pathway. 114

Molecular Immunotherapy Induction of T-cell immunity against EpCam (Epithelial Cell adhesion molecule) Oliver Schmetzer in cooperation with T. Kammertöns (MDC Antonio Pezzutto group of Molecular Immunology and Gene Therapy), P. Schlag (MDC group of surgical oncology) and K. Falk and O. Rötzschke (MDC group of cellular immunology of autoimmune reactions).

Some patients with colorectal cancer develop an immune response against peptides of the epithelial adhesion molecule EpCam, which is overexpressed in human adenocarcinomas. A sensitive ELISA assay for the screening of naturally occurring autoantibodies against EpCam, as well as circulating EpCam protein, has been established. Induction of autoantibodies against EpCam is an infrequent event occurring only in a minority of patients with adenocarcinomas (cooperation with P. Schlag). Some patients have circulating CD4 positive T cells that recognize MHC-II binding EpCam peptides. Since multimere peptides of MHC-II epitopes can amplify the natural occurring immune response, we are evaluating 4-mer and 16-mer EpCam multimeres in cooperation with K. Falk and O. Rötzschke. Transgenic mice expressing human Ep- Our group focuses on four topics: 1) Use of dendritic cells to Cam have been generated for use in preclinical vaccination induce tumor-specific immune response and identification of experiments (cooperation with T. Kammertöns, and Th. Blan- target antigens for immunotherapy by mass spectrometry ana- kenstein.) A clinical vaccination study in patients with lysis of MHC-bound peptides isolated from tumor cells; 2) EpCam-positive adenocarcinomas is in preparation. Analysis of the natural immunity against the adenocarci- noma-associated EpCAM antigen in cancer patients and vaccination in EpCAM transgenic mice; 3) Evaluation of gene DNA Vaccination transfer and DNA vaccination for induction of tumor specific Jörg Westermann, Tam Nguyen Hoai in cooperation with immunity; and 4) Evaluation of graft versus leukemia effects U. Höpken and M. Lipp (MDC group of Differentiation and in murine models of bone marrow transplantation. growth control in lymphocyte development).

In order to increase the delivery of genes to antigen presen- Use of dendritic cells for the induction ting cells, we have evaluated targeting of DNA-Polyethyleni- of anti-leukemic immune response mine-Mannose complexes to dendritic cells via the mannose Jörg Westermann, Monika Schwarz, and Tuan Duc Nguyen receptor. We have found that the cytokine Flt-3 Ligand in cooperation with E-M. Müller and Albrecht Otto (MDC (which can induce in vivo expansion and recruitment of group of Proteomics). dendritic cells) used together with DNA vaccination is not able to induce a TH1 polarized immune response. In coope- We have shown that T cells from both normal individuals and ration with the MDC group of M. Lipp (Molecular Tumor chronic myeloid leukemia (CML) patients can specifically Genetics), we are exploring the possibility of recruiting im- recognize the bcr-abl fusion peptide that is characteristic of mune cells at the vaccine site by inserting DNA sequences CML. To boost the leukemia specific immune response in coding for specific chemokine/chemokine receptors during patients, we perform clinical vaccination studies using in DNA vaccination. vitro-generated, bcr-abl positive dendritic cells (DC). Using tandem mass spectrometry (MS/MS) on peptides that are naturally presented and eluted from MHC-I molecules of leuke- Exploitment of the Graft versus Leukemia effect in mic cells, we have identified peptides that appear to identify the context of allogeneic stem cell transplantation. tumor associated antigens and are potential candidates for the Corinna Leng and Markus Y. Mapara development of vaccination strategies (cooperation project with E-M. Müller and A. Otto, (MDC Group of Proteomics). Allogeneic bone marrow/stem cell transplantation is currently Two HLA-A3 binding peptides from the LMO-4 and the the only curative treatment option for a number of malignant LYL-1 proteins appear to be particularly interesting. LMO-4 diseases of the blood system. The tumor responses achieved is the most recently identified member of a gene family of by this therapy depend to a significant extent on an immuno- “Lim-only” transcription factors that has been reported to be logically mediated graft-versus-leukemia (GVL) response in increased in breast cancer, prostate cancer, and some acute which immune effector cells of the donor (primarily T lym- T-cell leukemia cells. LYL-1 belongs to a subgroup of Helix- phocytes and to a lesser extent Natural Killer (NK) cells) Loop-Helix proteins that have been claimed to play a role in recognize the recipient as “foreign” and attack the recipient’s the development of acute T-cell leukemia. The technique for tumor cells (e.g. leukemia or lymphoma cells). Its widespread analysis of MHC-bound peptides by mass spectrometry is application, however, has been precluded by the development being extended to other cancer types. of graft-versus-host-disease (GVHD), which can lead to 115

A B C

isotype control anti-I-Ab, CD IIc anti-I-Ab, CD IIc PBS PBS FLT-3L

DC expansion induced by flt-3L Immunohistological staining of cryosections from the spleen of mice treated 12 days s.c. with PBS (B) or flt-3L (C). Tissue sections were stained with anti-I-Ab (anti-MHC class II, brown), anti-CD11c (red) or isotype control antibody (A). Flt-3L treated animals have high numbers of CD11c+MHC II+DC in their spleen

severe damage to the gut, liver, and skin. Our main research Richter G., M. Hayden-Ledbetter, M. Irgang, JA Ledbetter, J. interest in the context of allogeneic bone marrow transplanta- Westermann, I. Körner, K. Daemen, EA Clark, A. Aicher, A. tion (BMT) is to design strategies to separate graft-versus- Pezzutto. (2001). Tumor necrosis factor-alpha regulates the host-disease and graft-versus-leukemia reactions focusing on expression of inducible costimulator receptor ligand on three aspects: 1) modulation of T – APC interaction; 2) study- CD34(+) progenitor cells during differentiation into antigen ing the relevance of tumor associated antigens in GVL presenting cells. J. Biol. Chem. 276: 45686-45693. reaction in mixed chimeras, and 3) modulating the inflammatory response after bone marrow transplantation. Structure of the Group

Selected Publications Group Leader Prof. Dr. Antonio Pezzutto Westermann J., C. Schlimper, G. Richter, J. Mohm, B. Dörken A. Pezzutto. (2004). T cell recognition of bcr/abl in healthy Scientists donors and in patients with chronic myeloid leukemia. Brit. J. Dr. Jörg Westermann Hematol. in press. Dr. Markus Mapara Dr. Corinna Leng Westermann J., T. Nguyen-Hoai, A. Mollweide, G. Richter, Dr. Tam Nguyen-Hoai O. Schmetzer, H-M Kim, Th. Blankenstein, B. Dörken, A. Pezzutto. (2004). FLT-3 Ligand as adjuvant for DNA vacci- Graduate and Undergraduate Students nation augments immune responses but does not skew TH1/ Oliver Schmetzer TH-2 polarization. Gene Ther. in press. Mark Schnitzler Monika Schwarz Westermann J., J. Kopp, I. Körner, S. Kurz, M. Zenke, B. Tuan Duc Nguyen Dörken, A. Pezzutto. (2003).Cryopreservation of mature, monocyte-derived dendritic cells: DC maintain their pheno- Technical Assistants type and functional properties. Cancer Immunol Immunother, Daniela Tabor 52: 194-198 Simone Lusatis Sunda Jeske Mapara M.Y., Kim,YM, Marx JE, Sykes M (2003). DLI- mediated GVL effects in mixed chimeras established with a nonmyeloablative conditioning regimen: extinction of GVL effects following conversion to full donor chimerism. Trans- plantation 76:297-305 116

Molecular Immunology and Gene CTL generation. Even though necessary for CTL generation, Therapy CD4+ T cells did not need to express IL-4 or IL-4R. Surpri- singly, CTL generation required IL-4 but not IL-4R expression by CD8+ T cells. Since IL-4 (i) was expressed by naive Thomas Blankenstein CD8+ T cells within 24 h after antigen-encounter, (ii) IL-4 induced DC maturation and (iii) CTL development was impaired in T cell-reconstituted IL-4R–/– mice, CD8+ T cell-derived IL-4 appears to act on DCs. We conclude that CD4+ and CD8+ T cells provide different signals for DC activation during CTL generation.

Chemical carcinogenesis is inhibited by an IFNg-receptor-dependent foreign body reaction

The foreign body reaction is one of the oldest host defense mechanisms against tissue damage which involves inflammation, scarring, and encapsulation. The chemical carcinogen methylcholanthrene (MCA) induces fibrosarcoma and tissue damage in parallel at the injection site. Tumor development by MCA, but not p53-deficiency, is increased in interferon- receptor (IFNR) deficient mice. In the absence of IFNR, In the last two years, the group made several discoveries rela- MCA diffusion and DNA damage of surrounding cells are in- ted to the question of how the immune system rejects tumors. creased. Locally produced IFN induces the formation of a Importantly, the role of the tumor stroma as a target for im- fibrotic capsule. Encapsulated MCA can persist virtually life- mune mediated tumor rejection was investigated. Additio- long in mice without inducing tumors. Together, the foreign nally, a new mechanism was discovered showing that chemi- body reaction against MCA prevents malignant transforma- cal carcinogenesis can be spontaneously controlled in an tion, probably by reducing DNA damage, and it is more effi- immunological manner. Examples of the group’s activities cient in the presence of IFNR. This indicates that inflamma- during the report period are given below. tion and scarring, both suspected to contribute to malignancy, are protective during chemical carcinogenesis.

Tumor Rejection by Disturbing Tumor Stroma Cell Interactions CD8+ effector T cells reject tumors by direct antigen recognition but indirect action on host cells The stroma of solid tumors is a complex network of different cell types. Little is known about the stroma cell interactions in CD8+ effector T cells recognize malignant cells by monitor- a permissive or hostile tumor environment. We analyzed such ing their surface for the presence of tumor-derived peptides interactions in growing tumors and during their rejection. bound to major histocompatibility class (MHC) I molecules. Tumor infiltrating macrophages (TIM) require T cells to be- In addition, tumor-derived antigens can be cross-presented to come effector cells but, in growing tumors, are committed to CD8+ effector T cells by antigen-presenting cells (APCs). produce IL-10. Following T cell inactivation, TIM immedia- Interferon- (IFN) production by CD8+ T cells is often criti- tely switch to Interferon- (IFN) production and the tumor cal for tumor rejection. However, it remained unclear as to vasculature is destroyed in an IFN-receptor-dependent fashion. These events precede hemorrhagic necrosis and residual tumor cell elimination by T cells. Together, T cells suppress An IFN-supported foreign body reaction protects from chemical carcinogenesis their environment through regulation of macrophage function (methylcholanthrene). Encapsulated MCA persists within fibrotic area in long-term and tumor rejection can be induced by disturbing the tumor tumor-free mice. Shown is the staining with mAb ER-TR7 of a muscle section of a tumor-free IFN-R-WT mouse injected more than a year ago with MCA. The inlet figure stroma network. shows a piece of encapsulated MCA at higher magnification. Data are from Qin et al. (2002).

Generation of tumor-associated cytotoxic T lymphocytes requires IL-4 from CD8+ T cells

Activation of tumor-associated CD8+ cytotoxic T lymphocytes (CTLs) often requires antigen re-presentation, e.g. by dendritic cells (DCs), and CD4+ T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin-4 (IL-4) during the immunization but not effector phase. In order to determine the source and target cells of IL- 4, we performed adoptive T cell transfers using CD4+ and CD8+ T cells from IL-4–/– and IL-4R–/– mice and analyzed 117

whether (i) CD8+ T cells secrete IFN in response to antigen cal requirement of IFN-mediated angiostasis for tumor recognition on tumor cells or APCs and (ii) whether IFN rejection by CD8+ T cells. Cancer Res. 63: 4095-4100. mediates its anti-tumor effect by acting on host or tumor cells. We showed that CD8+ effector T cells can reject tumors in Schüler, T., Körnig, S. and Blankenstein, Th. (2003). Tumor bone marrow (BM) chimeric mice incapable of cross-presen- rejection by modulation of tumor stroma fibroblasts. J. Exp. ting antigen by BM-derived APCs and that tumor rejection Med. 198: 1487-1493. required host cells to express IFN receptor. Together, CD8+ effector T cells recognize antigen directly on tumor cells Qin, Z., Kim, H.-J., Hemme, J. and Blankenstein, Th. (2002). which is sufficient to reject tumors by IFN acting on host Chemical carcinogenesis is inhibited by an IFN-receptor-de- cells. pendent foreign body reaction. J. Exp. Med. 195: 1479-1490.

Ibe, S.*, Qin, Z.*, Schüler, T., Preiss, S. and Blankenstein, Th. A Critical Requirement of Interferon Gamma- (2001). Tumor rejection by disturbing tumor stroma cell inter- Mediated Angiostasis for Tumor Rejection by CD8+ actions. J. Exp. Med. 194: 1549-1560. (*equal contribution) T Cells Schüler, T., Kammertöns, T., Preiss, S., Debs, P., Noben- It is thought that tumor rejection by CD8+ T cell effectors is Trauth, N. and Blankenstein, Th. (2001). Generation of primarily mediated by direct killing. We show that rejection tumor-associated cytotoxic T lymphocytes requires IL-4 from of different tumors (fibrosarcoma, ras-transformed fibrob- CD8+ T cells. J. Exp. Med. 194: 1767-1775. lasts, colon carcinoma, plasmacytoma) by CD8+ T cells is always preceded by inhibition of tumor-induced angiogenesis. Angiostasis and tumor rejection were observed in perfo- Structure of the Group rin, but not in IFN-deficient mice. Furthermore, adoptive transfer of tumor-specific CD8+ T cells from IFN-competent Group Leader mice inhibited angiogenesis of lung metastases in comparison Prof. Dr. Thomas Blankenstein to those from IFN-gene deficient mice. Together with our previous findings, we conclude that IFN dependent anti- Scientists angiogenesis is a general mechanism involved in tumor rejec- Dr. Jehad Charo tion by CD4+ and CD8+ T cell effectors. Dr. Thomas Kammertöns Dr. Liang-Ping Li Dr. Susanne Preiß* Tumor rejection by modulation of tumor stromal Dr. Zhihai Qin fibroblasts Dr. Gerald Willimsky Dr. Jan Schmollinger* IL-4-secreting tumors are rejected in mice, an effect that is Dr. Thomas Schüler* thought to be immune-mediated. However, solid tumors are Dr. Hye-Jung Kim* embedded in a stroma that often contains tumor-promoting fibroblasts, a cell population whose function is also affected Graduate and Undergraduate Students by IL-4. We showed that IL-4-secreting tumors grew undimi- Christian Buschow nished in IL-4 receptor-deficient (IL-4R–/–) mice. In IL-4R+/+ Boris Engels mice, they were long-term suppressed in the absence of T Monika Gladow cells but complete rejection required T cells, compatible with Christoph Lampert the assumption that hematopoietic cells needed to respond to IL-4. Surprisingly, bone marrow (BM) chimeric mice revea- Technical assistants led that IL-4R expression exclusively on non-BM-derived Denise Barthel cells was sufficient for tumor rejection. Fibroblasts in the Angelika Gärtner tumor stroma were identified as a target cell type for IL-4, Martina Grabbert since they accumulated in IL-4-secreting tumors and dis- Nahid Hakiy played an activated phenotype. Additionally, co-injection of Sabrina Horn IL-4R+/+, but not of IL-4R–/– , fibroblasts was sufficient for Stefanie Kupsch the rejection of IL-4-secreting tumors in IL-4R–/– mice. Our Irmgard Küttner data demonstrate a novel mechanism by which IL-4 contri- Tanja Specowiak butes to tumor rejection and show that the targeted modula- Petra Straub tion of tumor-associated fibroblasts can be sufficient for Ralf Willebrand* tumor rejection. Christel Westen

Secretariat Selected Publications Sylvia Klahn

Qin, Z., Schwartzkopff, J., Pradera, F., Kammertöns, T., * part of the period reported Seliger, B., Pircher, H. and Blankenstein, Th. (2003). A criti- 118

Molecular Cell Biology and In a further project, we developed and analyzed recombinant Gene Therapy adenoviruses as gene transfer vectors. We evaluated the therapeutic potential of these vectors in animal experiments and analyzed possible reasons for side effects in clinical studies. Wolfgang Uckert We showed complement activation in isolated plasma of patients that harbored antibodies directed against adenoviral antigens after challenge with recombinant adenoviruses. Further- more, we found an increased specific affinity of the commonly used adenovirus serotype 5 (Ad5) and human erythrocytes leading to hemagglutination. This phenomenon is of importance with regard to the calculation of organ-specific gene transfer efficacies and may be linked to other patho- physiolocial reactions. Moreover, we showed that the use of constitutive nonregulated promoters, that are commonly used in adenoviral vectors, lead to a metabolic overload of transgenic cells.

Selected Publications

B. Engels, H. Cam, T. Schüler, S. Indraccolo, M. Gladow, C. Baum, T. Blankenstein, W. Uckert. Retroviral vectors for The group addresses questions related to the optimization of high-level transgene expression in T lymphocytes. Hum. viral vectors for gene transfer and the generation and transfer Gene Ther. 14: 1155-1168 (2003). of genetically modified T cells in tumor therapy. G. Cichon, S. Boeckh-Herwig, D. Kümin, C. Hoffmann, H. Retroviral vectors derived from Moloney murine leukemia H. Schmidt, E. Wehnes, W. Hänsch, U. Schneider, U. Eck- virus are commonly used to transfer foreign genetic informa- hardt, R. Burger, P. Pring-Akerblom. Titer determination of tion into T cells. We showed that retroviral vectors carrying Ad5 in blood: a cautionary note. Gene Ther. 10: 1012-1017 the envelope of murine leukemia virus 10A1 more efficiently (2003). transduce human primary T cells in comparison to vectors pseudotyped with the envelope of other retroviruses. How- K. Lange, W. Uckert, T. Blankenstein, R. Nadrowitz, C. Bitt- ever, when using these vectors, the expression level of trans- ner, J.-C. Renauld, J. van Snick, A. C. Feller, H. Merz. Over- ferred genes is often unsatisfactory. We analyzed different expression of NPM-ALK induces different types of malignant cis-regulatory elements and constructed retroviral vectors lymphomas in IL-9 transgenic mice. Oncogene 22: 517-527 (MP71) that yielded an up to 75-fold increase of transgene (2003). expression in T cells in comparison with those elements used in murine leukemia virus based vectors. P. Löser, C. Hoffmann, G. W. Both, W. Uckert, M. Hillgen- berger. Construction, rescue, and characterization of vectors Using an MP71 vector, the and chains of a T cell receptor derived from ovine atadenovirus. J. Virol. 77: 11941-11951 (TCR), that recognizes a tumor-associated antigen on renal (2003). cell carcinoma cells, was transferred into T cells. The TCR molecule was expressed on the cell surface of human primary M. Mühlebach, I. Schmitt, S. Steidl, J. Stitz, M. Schweizer, T. T cells of different donors. TCR gene-modified T cells could Blankenstein, C. Cichutek, W. Uckert. Transduction efficien- be stimulated in an antigen-specific, HLA-restricted fashion cies of MLV-, but not of HIV-1-vectors are pseudotype- and led to the recognition and lysis of renal cell carcinoma dependent on human primary T lymphocytes. J. Mol. Med. cells, comparable to T cell clones originally isolated from the 81: 801-810 (2003). tumor. Thus, the redirection of T cells by TCR gene transfer could greatly facilitate adoptive therapy. Structure of the Group However, little is known about how two TCRs on one T cell influence antigen recognition, specificity, and tumor cell ly- Group Leader sis. Therefore, we have generated a mouse model of double Prof. Dr. Wolfgang Uckert TCR T cells (OT-I x P14), specific for ovalbumin (ova) and LCMV glycoprotein 33 (gp33). These cells retain both speci- Scientists ficities and can be activated by their respective cognate pept- Dr. Günter Cichon ide. Adoptively transferred double TCR T cells suppress the Dr. Susanne Herwig growth of both B16-ova and B16-gp33 murine melanoma Dr. Niklas Köhler cells, regardless of the peptide used for in vitro T cell activa- Dr. Peter Löser tion. 119

Graduate and Undergraduate Students Petra Biese Boris Engels Monika Gladow Corinna Hofmann Simone Reuß Daniel Sommermeyer Xiaoqing Wu

Technical Assistants Uta Fischer Martina Grabbert Kordelia Hummel Irmgard Küttner Heidrun Peter 120

Molecular and Cell Biology of the kinetics of GATA-1 and c-Myb expression. We show that Hematopoietic Cells c-Myb expression is high in progenitors and effectively downregulated when GATA-1 is induced and cells differentiate into erythrocytes (Bartunek et al., 2003; in collaboration Martin Zenke with M. Dvorak, IMG, Prague). We also found that GATA-1 regulates c-Myb through binding to two GATA-1 sites in the c-myb promoter and this requires FOG-1. Thus, our study provides a direct molecular link between GATA-1 activity and c-myb expression during terminal red cell differentiation.

Transcriptional profiling by DNA microarrays identifies Id2 function in dendritic cell development Hacker, C., Ju, X.-S., Hieronymus, T., Kirsch, R. D., Kurz, S. M., Jorgas, T., Knespel, S., Euler, U.

Dendritic cells (DC) are professional antigen presenting cells that play key roles in antigen specific T cell responses and have been implicated in determining the balance between immunity and tolerance. DC originate from hematopoietic progenitor cells and occur throughout the organism, both in lymphoid and nonlymphoid tissues, and are specialized for Hematopoietic cells develop from a population of hemato- uptake, transport, processing, and presentation of antigens. poietic stem cells in bone marrow through multiple steps of Unique DC subclasses have been identified that differ in lineage commitment and differentiation. The focus of re- phenotype, function, activation state, and location but their search of this group is the molecular and cell biology of two relationship and developmental origins have remained un- hematopoietic cell types: red blood cells and antigen presen- clear or controversial. ting dendritic cells. Their development from stem/progenitor cells is studied in human cells and, in experimental model To study DC development, in vitro systems for differentiation systems, in hematopoietic cells of mouse and chicken. of human and mouse DC from hematopoietic stem/progenitor Emphasis is put on the identification of genes that determine cells were developed (Hacker et al., 2003; Ju et al., 2003; lineage fate and that will eventually allow to directly influ- Hieronymus et al., submitted). In these culture systems, cells ence cell fate and to generate cells with wanted properties. are grown with a stem cell factor/cytokine cocktail that main- tains the progenitor phenotype and cells are induced to undergo synchronous differentiation into DC by administration bFGF signaling, Myb, and GATA-1 transcription of GM-CSF and IL-4. Transcriptional profiling with microar- factors in red cell development rays was used to determine the transcription factor repertoire Bartunek, P. and Blendinger, G. of DC. This study identified the inhibitory helix-loop-helix transcription factor Id2 as one of the most prominently in- Red blood cells represent one of the most abundant speciali- creased factors during DC development in vitro (Hacker et zed cell types in vertebrate organisms. They develop from al., 2003). By studying Id2-/- mice we demonstrate that Id2 is hematopoietic stem cells through successive steps of differen- crucial for development of distinct DC subsets in vivo. Id2-/- tiation, including progenitors that are already committed to mice lack Langerhans cells (LC), the cutaneous contingent of the red cell lineage. We have now identified an erythroid pro- DC, and a specific splenic DC subset. TGF-/- mice also lack genitor that is critically dependent on bFGF and requires LC and we show that TGF acts upstream of Id2 and induces expression of v-Myb for sustained proliferation in vitro Id2 expression. (Bartunek et al., 2002; in collaboration with M. Dvorak, IMG, Prague). In the presence of bFGF such v-Myb cells are com- Id2-/- mice have higher B cell numbers than Id2+/+ mice and pletely blocked in their ability to differentiate and exhibit an we found that Id2 represses B cell genes in DC (Hacker et al., exceptionally high proliferative potential and long lifespan in 2003). Thus, a model presents itself where the relative vitro. In the absence of bFGF cells effectively differentiate expression levels of Id2 and activating HLH factor, such as into mature erythrocytes, irrespective of constitutive and E2A, determine lineage choice by affecting the propensity of elevated levels of v-Myb. Our studies suggest that bFGF, in such a common progenitor to develop into B cells or DC: low cooperation with Myb protein, represents an important factor or no expression of Id2 and high expression of E2A supports for determining erythroid lineage choice. B cell development while high expression of Id2 blunts E2A activity and B cell development, and thus allows differentia- Our previous work demonstrated that GATA-1 and GATA-2 tion into DC. transcription factors are important for red cell development (Briegel et al., Genes & Dev. 7, 1097-1109, 1993; Briegel et al., Development 122, 3839-3850, 1996; and references therein). We have employed a culture system that faithfully recapitulates red blood cell differentiation in vitro, to follow 121

ProgenitorDC CD+TNF I CD117, c-kit A CD113, AC133 B CD135, Flt3

II CD1b CD11c CD40 CD54, ICAM-1

III CD16, FcR CD206, mannose receptor IV CD1a CD11b CD80 CD83 CDw197, CCR7 CD205, DEC205 CD208, DC-LAMP

(A) Hierarchical cluster analysis of DNA microarray data of DC progenitor cells (progenitor) and differentiated DC (DC); DC+TNF, differentiated DC treated with TNF for an additional 16 hours. Green, low expression; black, intermediate expression; red, high expression (for details see Hacker et al., 2003). (B) Id2-/- mice lack Langerhans cells. Immunofluorescence analysis of epidermis of Id2-/- and Id2+/+ mice for MHC class II expression (top panel). Lower panel, phase contrast images of the respective fields in top panel (Hacker et al., 2003).

Gene transfer into antigen presenting dendritic Gust, T. C., Diebold, S. S., Cotten, M. and Zenke, M. (2004) cells (DC) RNA containing adenovirus/polyethylenimine transfer com- Gust, T. C. and Schröder, D. plexes effectively transduce dendritic cells and induce antigen-specific T cell responses. J. Gene Med., 6, 464–470. Given their unique properties in antigen specific T cell activation, DC represent a particularly attractive cell type for use Hacker, C., Kirsch, R. D., Ju, X.-S., Hieronymus, T., Gust, T. in immunotherapy of diseases. Following our interest in ge- C., Kuhl, C., Jorgas, T., Kurz, S. M., Rose-John, S., Yokota, nerating gene modified DC by receptor mediated endocytosis Y., and Zenke, M. (2003). Transcriptional profiling identifies (Diebold et al., Hum. Gene Ther. 10,775-786, 1999; Diebold Id2 function in dendritic cell development. Nat. Immunol. 4, et al., Gene Ther. 8, 487-493, 2001), we have now further 380-386. developed our gene delivery systems for DC. DC are postmitotic and notoriously difficult to transfect with DNA. Ju, X.-S., Hacker, C., Madruga, J., Kurz, S. M., Knespel, S., Accordingly, adenovirus polyethylenimine (Ad/PEI) RNA Blendinger, G., Rose-John, S., and Zenke, M. (2003). transfer complexes were generated that contain adenovirus Towards determining the differentiation program of antigen particles as ligand and in vitro transcribed RNA condensed by presenting dendritic cells by transcriptional profiling with PEI (Gust et al., 2004). These Ad/PEI/RNA complexes were DNA microarrays. Eur. J. Cell Biol., 82, 75-86. found to be more effective in transducing DC and in inducing antigen specific T cell responses than DNA containing com- Westermann J., J. Kopp, I. Körner, S. Kurz, M. Zenke, B. plexes. This is because transfected RNA readily reaches the Dörken, A. Pezzutto. (2003). Cryopreservation of mature, cytosol and is translated, thus obviating the need for nuclear monocyte-derived dendritic cells: DC maintain their pheno- translocation. type and functional properties. Cancer Immunol Immunother, 52: 194-198

Selected Publications Bartunek, P., Pajer, P., Karafiat, V., Blendinger, G., Dvorak, M. and Zenke, M. (2002). bFGF signalling and Myb coope- Bartunek, P., Kralova, J., Blendinger, G., Dvorak, M. and rate in sustained growth of primitive erythroid progenitors. Zenke, M. (2003). GATA-1 and c-myb crosstalk during red Oncogene 21, 400-410. blood cell differentiation through GATA-1 binding sites in the c-myb promoter. Oncogene 22, 1927-1935. 122

Structure of the Group

Group Leader Dr. Martin Zenke

Scientists Dr. Christine Hacker* Dr. Thomas Hieronymus Dr. Xin-Sheng Ju Dr. Ralf D. Kirsch* Dr. Ki-Ryang Koh

Graduate Students Mykola Goncharenko Tatjana C. Gust Thorsten Jorgas Steffen M. Kurz* Britt Lemke

Diploma Students Uta Euler* Dirk Schröder* Stefanie Strobach*

Visiting Scientists Dr. Petr Bartunek Ivonne Gamper* Susanne Günther

Technical Assistants Gitta Blendinger Siegne Knespel

Secretariat Petra Haink Patricia Podlatis**

* part of the time reported ** part-time 123

Cellular Immunology 2) Control of autoimmune reactions of Autoimmune Reactions Several mechanisms have been described which potentially allow the control of autoreactive T cells. Besides direct (or Kirsten Falk “suicidal”) mechanisms such as induction of ‘high-zone tole- Olaf Rötzschke rance’, indirect control mechanisms are most promising. Indi- rect control is mainly accomplished by regulatory T cells, which, upon antigen-specific activation, ‘silence’ or eliminate other activated immune cells in their vicinity. CD25+ CD4+ T cells have recently been identified to be one of the subpopulations responsible for this effect. Another subset are IL10 producing CD25– CD4+ T cells. Both subsets are currently under investigation. Some of the specific experimental tools employed in these studies are T cell epitope oligomers (repe- titive T cell antigens) and the heat shock protein gp96, which had been previously found to be effective tolerance inducers in vivo. Primary goals of these studies are the exploration of ways allowing for a specific recruitment or inactivation of these cells for the treatment of autoimmune diseases and cancer, respectively, the identification of key genes responsible for differentiation and maintenance of the suppressor status of regulatory T cells, and the characterization of the functional The driving force in the progression of autoimmune diseases role of regulatory T cell subsets. are the autoreactive T cells. In most cases, these autoreactive T cells are CD4+ T cells that have escaped the tolerance control mechanisms of the immune system. While they are 3) Selective activation of auto-reactive CD4 responsible for the induction of diseases, such as multiple effector T cells in tumour model systems sclerosis or diabetes they can be beneficial when recruited in the context of tumor immunotherapies. The group is currently While in autoimmune diseases the action of autoreactive interested in three basic problems related to the activation and CD4+ T cells can be fatal, it can be beneficial in the context of control of these cells: tumour-immunotherapies. In contrast to other therapies, the damage inflicted by these cells is very specific and restricted to the tissue expressing the autoantigen. Furthermore, the im- 1) Environmental factors for the induction of mune response of autoreactive CD4+ T is usually chronic and autoimmune reactions often leads to the recruitment of other immune cells, such as CD8+ CTL or B cells, which support or continue the tissue- It is already well established that genetic factors, such as specific removal of cells. In our group, initial tests are being expression of certain allelic forms of MHC class II molecules, carried out in which the capacity of autoreactive CD4+ T cells play an important role in autoimmune diseases. At least is tested with samples from tumour patients and with experi- equally important for the induction of these diseases are envi- mental mouse model systems. The trials employ antigens with ronmental influences. Up to now, however, these environ- enhanced immunogenicity, such as epitope oligomers or lipo- mental factors are largely unknown. Recent experiments in peptides, and include the construction of inducible animal our group demonstrated that certain small molecular compo- model systems (TET system). Results obtained within the two unds are able to trigger the exchange of peptide antigens on other focal points of our research (small molecular ligand the surface of activated antigen presenting cells. These anti- exchange catalysts and immuneregulation) are implemented gens include autoantigens (peptides and proteins), the target in these trials to achieve additional leverage by generating structure of autoreactive T cells. Studies by other groups have proinflammatory environments promoting tumor-specific shown that peptides derived from these autoantigens can in- immune responses. duce fatal autoimmune reactions when loaded onto activated dendritic cells. By catalysing this process, these small molecular compounds might, therefore, represent environmental Selected Publications risk factors which have not been considered previously. On the other hand, these compounds could also be applied in the- Falk, K., and O. Rotzschke. 2002. The final cut: how ERAP1 rapeutic settings. For instance, by mediating the transfer of trims MHC ligands to size. Nat. Immunol. 3, 1121-1122. tumor-specific antigens, they can enhance immune responses directed against the transformed tissue. The group is currently Rotzschke, O., J. M. Lau, M. Hofstatter, K. Falk, and J. L. defining the structural requirements of the compounds and in- Strominger. 2002. A pH-sensitive histidine residue as control vestigates their impact on several experimental autoimmune element for ligand release from HLA-DR molecules. Proc. and tumor model systems. Natl. Acad. Sci. USA 99, 16946-16950.

Falk, K., J.M. Lau, L. Santambrogio, V. Marin Esteban, F. Puentes, O. Rötzschke*, and. J.L. Strominger (2002). Ligand- 124

exchange of MHC class II proteins is triggered by H-bond donor groups of small molecules. J. Biol. Chem. 277, 2709- 2715 *corresponding author.

Steinekemeier, M., K. Falk, O. Rötzschke, A. Weishaupt, C. Schneider, K.V. Toyka, R. Gold, and J.L. Strominger (2001). Vaccination, prevention and treatment of experimental autoimmune neuritis (EAN) by an oligomerized T cell epitope. Proc. Natl. Acad. Sci. U S A 98, 13872.

Falk, K., Rotzschke, O., Santambrogio, L., Dorf, M.E., Brosnan, C., and Strominger, J.L., (2000). Induction and suppression of an autoimmune disease by oligomerized T cell epitopes: enhanced in vivo potency of encephalitogenic peptides. J. Exp. Med. 191, 717-730.

Structure of the Group

Group Leaders Dr. Kirsten Falk Dr. Olaf Rötzschke

Scientists Dr. Mireille Starke

Graduate and Undergraduate Students Eva Goldscheid Viviana Marin Esteban Markus Kleinewietfeld Fabiola Puentes Sabine Höpner Reiner Mailer

Technical Assistants Maria Hofstätter Sabrina Kleißle Kerstin Petsch 125

Experimental Pharmacology (EpCAM, E-cadherin, CEA). Their response rate towards clinically used drugs was 33% for 5-Fluorouracil, 100% for Irinotecan, and 57% for Oxaliplatin. In five patients who Iduna Fichtner received chemotherapy for synchronous metastases, the responsiveness of the malignancy correlated excellently with the responsiveness of the corresponding xenografts. All of the xenografts expressed the proliferation marker Ki67 and the nuclear enzyme Topoisomerase II. Sixty-four percent (64%) of the xenografts expressed the mutated tumour suppressor p53. Interestingly, the detection of K-ras mutations in codon 12 in 30% of the carcinomas coincided with a low response to Oxaliplatin.

These well-characterized models are useful tools for the preclinical development of novel therapeutic approaches and for investigating translational research aspects.

Procedure for in vivo gene transfer

The clinical use of gene therapeutic strategies for the treatment of cancer is still hampered by insufficient transfer Translational research, bridging the gap between fundamental efficiencies and specificities in the in vivo situation. We in- and clinical research, is the main research focus of our group. vestigated a non-viral gene transfection system utilizing lipo- Using preferably in vivo mouse models, we are interested in plexes. As an additional component, alkyl phospholipids investigations concerning novel therapeutic approaches for (APL) were included because of their known interference the treatment of cancer. Recent research dealt with three main with cellular membranes. The APL lipoplexes showed in vitro topics: the highest transfer efficiencies into colon carcinoma cells. The LacZ reporter gene expression in colon xenografts was • Characterization of patient-derived leukemias and colon equally intense as revealed after use of lipofectin. A suicide carcinomas for clinical relevance • Testing of in vivo procedures for gene therapy • Stem cell engraftment and proliferation

A Nude mouse bearing a patient-derived colon carcinoma xenograft B NOD/SCID mice xenotransplanted with a human leukemia Clinical relevance of xenotransplanted malignancies

It is essential that pre-clinical models used to predict the activity of novel therapeutic strategies for the treatment of cancer coincide in decisive parameters with the clinical phenotype. Therefore, we were interested in the establishment of xenografts directly from patient material into immunodeficient mice. In vivo transplantable lines were maintained in early passages and characterized in terms of their relation to the original samples.

We could show that, after several murine passages, acute lymphoblastic leukemias (ALL) still resembled the clinical A disease in terms of both immuno- and genotype but also in relation to chemoresponsiveness. ALL established from primarily diagnosed patients revealed a higher response to anti-leukemic drugs than leukemias from relapsed patients. The treatment success was conversely related to the expression of resistance markers (P-gp, LRP, MRP1) on the leukemic cells.

Studies performed in close cooperation with the Robert- Rössle-Cancer Clinics aimed to establish xenografts from colorectal carcinomas. Starting with 35 surgical samples, we were able to establish 15 transplantable lines in nude mice. The xenografts coincided with the original tumor both in histology and in the expression of tumour-associated markers B 126

therapy with the cytosine deaminase gene + 5-Fluorocytosine Zeisig, R., Ress, A., Fichtner, I., and Walther, W. (2003). in a murine colon carcinoma resulted in the highest tumor Lipoplexes with alkylphospholipid as new helper lipid for growth inhibition when APL containing lipoplexes were used efficient in vitro and in vivo gene transfer in tumor therapy. for gene transfer. Cancer Gene Therapy 10, 302-311.

Nitsche, A., Junghahn, I., Thulke, S., Aumann, J., Radonic, Stem cell engraftment and proliferation A., Fichtner, I., and Siegert, W. (2003). Interleukin-3 promotes proliferation and differentiation of human hematopoietic Continuing former studies, we were further interested in stem cells but reduces their repopulation potential in NOD/ methods for the controlling of the engraftment potential of SCID mice. Stem Cells 21, 236-244. haematopoietic stem cells. For this purpose, cord blood-derived CD34-positive progenitor cells were transplanted into Becker, M., Nitsche, A., Neumann, C., Aumann, J., Junghahn, highly immunodeficient NOD/SCID mice. In order to subse- I., and Fichtner, I. (2002). Sensitive PCR method for the quently monitor a successful engraftment, we developed a detection and real-time quantification of human cells in sensitive PCR method for the detection and real-time quanti- xenotransplantation systems. Brit. J. Cancer 87, 1328-1335. fication of human cells in xenotransplantation systems. This universally applicable method targets a 850-bp fragment of the alpha-satellite DNA on human chromosome 17. The Structure of the Group method allows for the detection of one human cell in 106 mouse cells and could monitor the engraftment rate of stem Group Leader cells in a time dependent fashion. Dr. Iduna Fichtner

A further study explored the influence of human interleukin-3 Scientists (IL-3) on the production of human cordblood derived haema- Dr. Michael Becker topoietic cells in NOD/SCID mice. The background for that Dr. Klaus Eckert study was the knowledge that not all haematopoietic cyto- Dr. Reiner Zeisig kines are cross-reactive between mice and human. In particu- Dr. Ilse Junghahn lar, IL-3 is highly species-specific but plays a crucial role in hematopoiesis. We used, as a source for human IL-3, a stably Graduate and Undergraduate Students transfected rat fibroblast cell line, which was co-transplanted Diana Behrens to NOD/SCID mice. Rat-IL-3 mice displayed a higher Claudia Keil engraftment of human haematopoietic cells in bone marrow, Lisa Selders spleen, and peripheral blood when compared with mice Annika Wulf bearing a mock-cotransplant. Successive experiments with long term bone marrow cultures or secondary transplantations Technical Assistants showed that IL-3 could also play a role in the depletion of Monika Becker haematopoietic stem cells from the chimeric bone marrow of Margit Lemm mice. Claudia Neumann Anne-Dorothe Tepke Ongoing studies with cordblood stem cells investigate the differential expression of adhesion and apoptosis related Secretariat markers after co-cultivation with tissue specific factors. Sylvia Schulz These experiments are the precondition for scheduled in vivo studies concerning the transdifferentiation potential of early haematopoietic progenitors.

Selected Publications

Fichtner, I., Slisow, W., Gill, J., Becker, M., Elbe, B., Hille- brand, T., and Bibby, M. (2004). Anticancer drug response and expression of molecular markers in early-passage xenotransplanted colon carcinomas. European J. Cancer, 40, 298- 307.

Fichtner, I., Paal, K., Borgmann, A., Badiali, L, Wurm, R, and Henze, G. (2003). Chemo- and radiation sensitivity of xeno- grafted acute lymphoblastic leukemias - correlation to the expression of multidrug resistance proteins. Anticancer Res. 23, 2657-2664. Function and Dysfunction of the Nervous System

Signalling Pathways in the Nervous System

Pathophysiological Mechanisms of Neurological and Psychiatric Disorders

Imaging of the Live Brain

Coordinators: Carmen Birchmeier Helmut Kettenmann 128

Function and Dysfunction of the Funktion und Dysfunktion des Nervous System Nervensystems

Carmen Birchmeier Carmen Birchmeier Helmut Kettenmann Helmut Kettenmann

A single neuron can contact many neuronal and glial cells. Ein einzelnes Neuron kann viele Neurone und Gliazellen The structure and function of the nervous system are thus kontaktieren, Struktur und Funktion des Nervensystems sind very complex. The understanding of the molecular mecha- daher sehr komplex. Wir verstehen bisher nur begrenzt die nisms used to establish and maintain neuronal circuits, or to molekularen Mechanismen, die die neuronalen Schaltkreise process and store information in such circuitry, is still limi- aufbauen und erhalten. Ebenso ist noch nicht bekannt, wie ted. Recent advances in methodology and the available Informationen in solchen Netzwerken verarbeitet und gespei- genome sequence of humans and model organisms now allow chert werden. Technologische Fortschritte und die Verfügbar- a more systematic molecular analysis and will accelerate the keit der Genomsequenz von Menschen wie Modellorganis- furthering of our knowledge. men erlauben nun eine systematischere Analyse und werden uns weitere Einblicke in die Funktionen des Gehirns ermög- The research program in Neuroscience at the MDC focuses lichen. on molecular and cellular aspects and relies on the use of molecular biology, biochemistry, genetics, immunocytoche- Das Forschungsprogramm Neurowissenschaften am MDC mistry, electrophysiology, and anatomy to understand the stellt die molekularen und zellulären Aspekte in den Vorder- formation and function of the nervous system. Particular grund. Es nutzt Ansätze der Molekularbiologie, Biochemie, emphasis is also devoted to the analysis of mechanisms that Genetik, Immunzytochemie, Elektrophysiologie und Anato- underlie neuropathological conditions. mie, um die Entwicklung und Funktion des Nervensystems zu verstehen. Ein besonderer Schwerpunkt liegt auf der Analyse Several of the recent findings of the department have impor- der Mechanismen von neuropathologischen Veränderungen. tant medical implications. Erich Wanker and his group study the aggregation of proteins as they occur in Alzheimer’s, Eine Anzahl neuer Ergebnisse aus dem Bereich hat wichtige Parkinson’s, or Huntington’s disease. Agents that prevent medizinische Implikationen. Erich Wanker und seine Gruppe these aggregates, also known as amyloid plaques, are expec- untersuchen die Aggregation von Proteinen, die eine wichtige ted to be beneficial for the treatment of these diseases. Using Rolle bei Alzheimer, Parkinson und Huntington Erkrankun- automated cell-based and cell-free screening assays, they gen spielen. Substanzen, die eine solche Aggregation verhin- recently identified such inhibitors and are currently testing dern, können den Verlauf dieser Erkrankungen positiv beein- their activity in animal models, i.e. transgenic flies and mice. flussen. In einem automatisierten Screening-Verfahren haben Compounds able to reduce aggregate formation and neuro- sie kürzlich solche Inhibitoren entdeckt und testen jetzt deren logical symptoms in vivo are candidate substances for Aktivität in Tiermodellen, wie zum Beispiel transgenen Flie- clinical trials for these neurodegenerative diseases (Ross et gen oder Mäusen. Substanzen, die die Aggregatbildung redu- al., Proc. Natl. Acad. Sci. USA 100, 1-3, 2002). zieren und neurologische Symptome lindern, sind potentielle Substanzen für klinische Studien (Ross et al., Proc. Natl. Stimulation of neuronal regeneration in patients with neuro- Acad. Sci. USA 100, 1-3, 2002). degenerative disease might be an interesting therapeutic tool for the future. Gerd Kempermann and Alistair Garratt aim to Die Stimulation neuronaler Regeneration bei Patienten mit understand the properties and behaviour of neuronal stem neurodegenerativen Erkrankungen könnte ein interessantes cells. Gerd Kempermann and his team could recently distin- therapeutisches Werkzeug der Zukunft sein. Gerd Kemper- guish stem cell populations in the adult hippocampus which mann und Alistair Garratt studieren die Eigenschaften und respond differently to physical activity or changes in environ- das Verhalten neuronaler Stammzellen. Gerd Kempermann mental stimulation (Kronenberg et al., J. Comp. Neurol. 467, und sein Team konnten kürzlich drei Populationen im 129

455-463, 2003). Alistair Garratt is analysing the function of erwachsenen Hippocampus unterscheiden, die unterschied- growth factors in the maintenance and differentiation of adult lich auf physische Aktivität oder Veränderung von Umwelt- neuronal stem cells. Cortical spreading depression is a slowly reizen reagieren (Kronenberg et al., J. Comp. Neurol. 467, propagating wave of neuronal depolarization in the mamma- 455-463, 2003). Alistair Garratt untersucht die Funktionen lian brain, and is thought to play a role in pathological condi- von Wachstumsfaktoren, die erwachsene neuronale Stamm- tions, for instance in migraine headaches. zellen stabilisieren und deren Differenzierung steuern. „Cor- tical spreading depression“ ist eine sich langsam ausbreitende Neuronal depolarization was found to be accompanied by Welle neuronaler Hemmungen im menschlichen Gehirn, und Ca2+ activity of glia, the cell type studied by Helmut Ketten- man vermutet, dass sie bei neurologischen Symptomen eine mann´s group. Waves of Ca2+ elevations spread within a glial Rolle spielen wie zum Beispiel bei Migräne. population of the brain, the astrocytes. Interestingly, the astrocyte Ca2+ wave penetrates a larger territory than the Diese neuronale Depolarisation ist begleitet von einer Kalzi- activity of neurones and by this represents a self-reliant phe- umaktivität der Gliazellen, die in Helmut Kettenmanns Labor nomenon (Peters et al., J. Neurosci. 23, 9888-9896, 2003). untersucht werden. Diese Kalziumerhöhungen breiten sich wellenartig in einer glialen Zellpopulation des Gehirns, den Christiane Alexander found that mutations in the OPA-1 gene Astrozyten, aus. Interessanterweise breitet sich die Kalzium- result in degeneration of retinal ganglion cells. Patients affec- welle der Astrozyten über einen größeren Bereich des Ge- ted by such a gene defect face a loss of vision starting within hirns aus als die Aktivität der Neurone (Peters et al., J. Neu- the first two decades of their life. Her research focuses on the rosci. 23, 9888-9896, 2003). functional role of this protein, which is distributed ubiquitously, but its malfunction leads to a primary retinal defect. Christiane Alexander konnte zeigen, dass Mutationen im OPA-1 Gen zu einer Degeneration der retinalen Ganglienzel- The sense of touch is not a single sense, since neuroscientists len führt. Patienten mit einem solchen Gendefekt verlieren distinguish the perception of texture, temperature, and pain. ihr Augenlicht innerhalb der ersten beiden Lebensjahrzehnte. Distinct neurons located in sensory ganglia innervate the skin Sie untersucht daher die funktionelle Bedeutung des Proteins, and relay this information to the central nervous system by das zwar in allen Zellen verbreitet ist, dessen Fehlfunktion je- projecting to the spinal cord. Neurons in the spinal cord doch primär die Retina betrifft. integrate and process the sensory information and transmit it to higher brain centres. Sensory neurons that detect stimuli Der Tastsinn ist nicht eine einzelne Wahrnehmung. Neuro- such as a light brush of the skin or intense, painful heat are wissenschaftler unterscheiden die Wahrnehmung von Textur, functionally distinct, but marker genes whose expression Temperatur und Schmerz. Definierte Neurone in den senso- distinguishes them were unknown. Gary Lewin´s group used rischen Ganglien innervieren die Haut und leiten ihre Infor- microarray techniques to identify a calcium channel that is mation an das Zentralnervensystem weiter, indem sie ins specifically expressed in sensory neurons that detect light Rückenmark projizieren. Die Neurone im Rückenmark inte- brush. This channel enhances the sensitivity of mechano- grieren und verarbeiten diese sensorische Information und receptive neurons and its expression is a unique marker to leiten sie in höhere Hirnzentren weiter. Sensorische Neuro- define this subtype of sensory neuron (Shin et al., Nature nen, die leichte Berührung der Haut erkennen, unterscheiden Neuroscience 6, 724-730, 2003). sich von denen, die schmerzvolle Hitzereize erkennen. Bisher konnten sie genetisch nicht unterschieden werden. Gary How neurons form precise and selective connections is a cen- Lewins Gruppe nutzte die Technik der Micro-Arrays, um tral question in developmental neurobiology. The growth einen Kalziumkanal zu finden, der nur in sensorischen Neu- cone of sensory neurons detects guidance cues that are neces- ronen exprimiert wird, die leichte Berührung erkennen. Die- sary for the innervation of the spinal cord. The cGMP mes- ser Kanal verstärkt die Sensitivität der mechanosensitiven senger pathway can convert repulsive pathfinding cues to at- Neuronen, und durch seine Expression kann dieser Subtyp tractive ones, and is therefore an important modulator of von sensorischen Neuronen erkannt werden (Shin et al., neuronal pathfinding. Fritz Rathjen and his collaborators cha- Nature Neuroscience 6, 724-730, 2003). racterized the significance of cGMP signalling in sensory innervation of the spinal cord. In mice lacking the gene enco- Wie Neurone präzise und selektive Verbindungen aufbauen, ding the cGMP-dependent protein kinase I, sensory axons fail ist eine zentrale Frage in der Entwicklungsneurobiologie. Der to bifurcate correctly at the entrance zone of the spinal cord. Wachstumskegel sensorischer Neurone erkennt Wegmerk- Instead they grow directly to the central canal in these mutant male, die notwendig sind, um das Rückenmark zu innervie- mice. As a result, the animals have lost their reflex response ren. Der cGMP Signalweg kann inhibitorische Signale in po- to pain (Schmidt et al., J Cell Biol. 159, 489-498, 2002). Dor- sitive verwandeln und ist daher ein wichtiger Modulator der sal horn neurons are known to be diverse, but nevertheless neuronalen Zielfindung. Fritz Rathjen und seine Mitarbeiter develop from only a few distinct postmitotic neuron types, untersuchen die Bedeutung des cGMP Signalweges für die which were recently defined. sensorische Innervation des Rückenmarks. In Mäusen, bei denen das Gen für die cGMP-abhängige Proteinkinase 1 aus- geschaltet ist, verzweigen sich die sensorischen Axone nicht mehr korrekt in der Eingangszone des Rückenmarks. Statt- dessen wachsen sie in diesen Mutanten direkt zum zentralen Kanal. Dies hat zur Folge, dass die Tiere eine verminderte Reflexantwort für Schmerz zeigen (Schmidt et al., J Cell 130

Carmen Birchmeier and her group identified an important Biol. 159, 489-498, 2002). Neurone des dorsalen Horns set- gene product, the homeodomain factor Lbx1, which distin- zen sich bekannterweise aus verschiedenen Populationen zu- guishes two major neuronal classes in the dorsal spinal cord sammen. Sie entwickeln sich jedoch, wie kürzlich gezeigt, and is an important determinant of their distinct differentia- aus wenigen Typen von postmitotischen Neuronen. tion programs. In Lbx1 mutant mice, highly abnormal differentiation of dorsal horn neurons is accompanied by altera- Carmen Birchmeier und ihre Gruppe identifizierten ein wich- tions in the innervation of the dorsal horn by cutaneous tiges Genprodukt, den Homeodomänfaktor Lbx1, durch des- sensory neurons. These changes in circuitry are accompanied sen Expression sich zwei wichtige neuronale Populationen im by deficits in sensory reflexes (Müller et al., Neuron 34, 551- dorsalen Rückenmark unterscheiden und der ein wichtiger 562, 2002). Stefan Britsch studies the genome-wide expres- Faktor für ihr Differenzierungsprogramm ist. In den Lbx mu- sion of genes during spinal cord development, and currently tanten Mäusen entwickeln sich die Neurone des dorsalen analyses the function of homeodomain factor Gbx1 in the Horns abnormal, und dies ist begleitet von einer veränderten development of spinal cord neurons. Innervation des dorsalen Horns durch die sensorischen Neu- rone der Haut. Diese veränderten neuronalen Verbindungen The Neuroscience research program has expanded considera- gehen mit defekten sensorischen Reflexen einher (Müller et bly during the reported period. The research groups of Erich al., Neuron 34, 551-562, 2002). Stefan Britsch untersucht die Wanker and Carmen Birchmeier joined the department, and Genomweite Expression von Genen während der Rücken- Gary Lewin, previously a junior group leader in the depart- marksentwicklung und studiert derzeit die Bedeutung des ment, received tenure and is now full professor at the Homeodomänfaktors Gbx1 während der Entwicklung der Humboldt University of Berlin. Since the completion of the Rückenmarksneurone. communication centre of the MDC, larger scientific conferences can be accommodated on the campus. One neuros- Im Berichtszeitraum wurde das Forschungsprogramm Neuro- cience conference that took place on the campus was the VI wissenschaften stark erweitert. Die Forschungsgruppen von European Meeting on Glial Cell Function in Health and Erich Wanker und Carmen Birchmeier sind zu dem Koordi- Disease in September 2003. It was attended by more than 500 nationsbereich gestoßen, und Gary Lewin, zuvor Nachwuchs- scientists from all over the world. gruppenleiter im Bereich, leitet nun eine etablierte Gruppe und wurde zum Professor an der Humboldt Universität zu Other activities were a meeting of the faculty and students of Berlin ernannt. Seit Eröffnung des Kommunikationszentrums the Neuroscience Department in 2003 at Chorin in Branden- am MDC ist es möglich, große Konferenzen am Standort burg, which was used to present and to discuss ongoing re- durchzuführen. Ein solches Ereignis war das „6th European search and foster interactions in a relaxed and informal envi- Meeting on Glial Cell Function in Health and Disease“ im ronment. This department meeting alternates with the Berlin September 2003, das mehr als 500 Wissenschaftler aus aller Neuroscience Forum that is attended by neuroscientists from Welt anzog. all the neuroscience institutes in Berlin, and that took place in Liebenwalde in 2002. A seminar series that is organized Zu den weiteren Aktivitäten gehörte ein gemeinsames Sym- jointly by all neuroscience faculty members was established posium aller Neurowissenschaftler des MDC, das 2003 in in 2003. It developed into a very successful lecture series that Chorin, Region Brandenburg, durchgeführt wurde und als is regularly attended by many scientists and students, both Forum für die Diskussion neuester wissenschaftlicher Ergeb- from the neuroscience as well as other MDC departments. nisse und zur Planung weiterer Zusammenarbeiten genutzt wurde. Dieses Department-Treffen wechselt sich mit dem Berlin Neuroscience Forum ab, bei dem Neurowissenschaft- ler aus allen Berliner Institutionen zusammenkommen. Der Koordinationsbereich etablierte im Jahr 2003 ein gemeinsames Vortragsprogramm, das für viele Wissenschaftler und Studenten sowohl im Neuro- als auch aus anderen Bereichen des MDC attraktiv ist. Signalling Pathways in the Nervous System

Pathophysiological Mechanisms of Neurological and Psychiatric Disorders

Imaging of the Live Brain

Mouse Genetics – Tools for the first neurogenic wave (E10.5 in the mouse), distinct types of Functional Analysis of Genes that neurons arise at stereotyped positions along the dorso-ventral axis in the developing spinal cord. Prior to the emergence of are Important for Development and postmitotic neurons, neuronal progenitor cells possess regio- Disease nal identities and distinct progenitor populations generate the different neuronal subtypes. Carmen Birchmeier In the dorsal portion of the spinal cord, progenitor cells are patterned by extrinsic signals. Members of the BMP and wnt families are expressed in or near the roof plate at the time dorsal progenitor domains are established. Six postmitotic neu- We are using mice as a model organism for the functional ana- ron types (named dorsal interneurons (dI) and numbered from lysis of genes important for embryonic development and di- 1 to 6 along the dorso-ventral axis) can be distinguished. We sease. Tools for molecular genetics are well established in showed that these six neuron types can be classified based on mice. Homologous recombination and embryonic stem cell the expression of the Lbx1 gene (Fig. 1): Class A neurons technology make it possible to introduce targeted deletions or (dI1-dI3) do not express Lbx1, arise in the dorsal portion of insertions into the mouse genome. A further development of the dorsal spinal cord, require dorsal signals for specification, the technique, the Cre-LoxP technology, allows for the intro- and settle in the deep dorsal horn. In contrast, class B neurons duction of subtle alterations, like point mutations or conditio- (dI4-dI6) express Lbx1, emerge independently from roof nal mutations, that are restricted to a particular cell lineage. plate signals in the ventral portion of the dorsal spinal cord, and most eventually settle in the ventral spinal cord and in the deep dorsal horn. Development of the spinal cord Thomas Müller, Henning Brohmann, Hagen Wende, Mathias During a later phase known as the second neurogenic wave Gierl, Hendrik Wildner, Dominique Bröhl (E12.5 in the mouse), most neurons born in the dorsal spinal cord express Lbx1 and, thus, have a class B character. We The dorsal horn of the spinal cord is the first central relay defined two late Lbx1+ neuronal subtypes (dILA and dILB) station for somatosensory perception. Interneurons and pro- (Fig. 2) that arise in a salt-and-pepper pattern and settle in the jection neurons in the dorsal horn integrate incoming sensory dorsal horn. Lbx1 not only delineates two major dorsal neuro- information and transmit this information to higher brain nal classes but also specifies the differentiation program of centers. The assembly of these complex neuronal circuits class B neurons. In Lbx1 mutant mice, early and late class B depends on the generation of functionally distinct types of neurons express transcription factors typical of class A neu- dorsal horn neurons. Physiological studies have defined many rons. Conversely, misexpression of Lbx1 in the chick spinal distinct populations of dorsal horn neurons, which (i) process cord suppresses the emergence of class A neurons and, in- sensory information associated with touch, pain, and heat per- stead, induces the generation of class B neurons (Fig. 3). The ceptions, (ii) modulate reflex-specific motoneuron output, abnormal differentiation of the dorsal horn neurons in Lbx1 and (iii) relay afferent sensory information to the brainstem mutants is accompanied by changes in the histology and cir- and the thalamus. Neurons with different physiological pro- cuitry of the spinal cord. Thus, Lbx1 is essential for the spe- perties are segregated in distinct laminae of the dorsal horn. cification and differentiation of class B neurons and suppres- The cascade of events that specifies the development of these ses the development of class A neurons (Müller et al, 2002). different neuronal subtypes is unclear and, thus, we are analyzing this process by the use of mouse genetics. We are systematically extending these studies and are analyzing the function of other genes in dorsal horn development.

The role of Lbx1 in spinal cord development Thomas Müller, Henning Brohmann The Neuregulin/ErbB signaling system Alistair Garratt, Thomas Müller, Cemil Özcelik, Simone Lier The dorsal part of the spinal cord gives rise to neurons that process and relay sensory information, whereas the ventral Neuregulin-1 is an EGF-like growth and differentiation factor part generates motoneurons and interneurons that coordinate that signals through tyrosine kinase receptors of the ErbB motor output. During an early developmental phase called the family. Two receptors, ErbB3 and ErbB4, bind Neuregulin-1 132

Fig. 1. Lbx1 marks class B neurons in the dorsal spinal cord at E10.5. Sections of E10.5 spinal cords were stained with antibodies against the markers shown above the panels (A- D). Data are summarized in E. Interneuron types dI1-6 are marked. Bar: 100 um.

Fig. 2. The late class B neuron types dILA and dILB arise from the entire Mash1+ progenitor domain in a salt-and-pepper pattern at E12.5. Sections of E12.5 spinal cords were stained with antibodies as shown above the panels. Isl1/2+ dI3 neurons have reached already their final location in the deep dorsal horn (arrow, left panel). Lbx1+ neurons arise from the entire Mash1+ progenitor domain and settle in upper layers of the dorsal horn. dILA and dILB neurons arise in a salt-and-pepper from the Mash1+ progenitor domain (middle and right panels). Bars: 100 um.

with high affinity and are expressed in distinct patterns during Role of the Neuregulin signaling system in heart development: ErbB3 is expressed in neural crest and glial development and function cells , whereas ErbB4 is expressed in the heart. A third recep- Alistair Garratt, Cemil Özcelik tor, ErbB2, is present ubiquitously and acts as an essential co- receptor. Accordingly, functional receptor complexes in vivo Mice with null-mutations in Neuregulin/ErbB2 display defi- are either ErbB2/3 or ErbB2/4 heteromers, depending on the cits in heart development, that causes embryonic lethality at organ or cell type. We have introduced null-mutations into mid-gestation. This genetic analysis revealed for the first time mouse Neuregulin-1, ErbB2 and ErbB3 genes. All of these a role of Neuregulin/ErbB2 in cardiomyocytes which is also mutations cause either embryonic or postnatal lethality. of interest for subsequent observations of side-effects of Therefore, we introduced more subtle mutations that elimi- ErbB2 antibody therapy used now in patients for tumor treat- nate particular isoforms of Neuregulin, or conditional muta- ment (a proportion of such patients develops cardiomyopa- tions in the ErbB2 receptor. Together, the analysis of these thies). To analyze whether ErbB2 has an essential role in adult mutants revealed diverse, essential functions of the Neuregu- heart function, we used the Cre-loxP technology to mutate lin/ErbB signaling system in the development of the heart, the ErbB2 specifically in cardiomyocytes. Such conditional mu- neural crest cells, and the peripheral nervous system. tant mice develop a severe dilated cardiomyopathy with signs of cardiac dysfunction appearing by the second postnatal 133

Structure of the Group

Group Leader Prof. Dr. Carmen Birchmeier

Scientists Dr. Thomas Müller Dr. Alistair Garratt* Dr. Stefan Britsch* Dr. Henning Brohmann Dr. Cemil Özcelik* Dr. Hagen Wende* Dr. Simone Lier* Dr. Elvira Rohde*

Fig. 3. Schematic diagram of dorsal spinal cord neurons and of the function of Lbx1 in Visiting Scientists their development. BMP and Shh, secreted factors that pattern the dorsal (D) and Prof. Dr. Margaret Martinez de la Torre* ventral (V) progenitor domains, are indicated at the left. The class A (green) and B (red) neuronal subtypes generated in the dorsal spinal cord are indicated. The positions along the dorso-ventral axis at which these neurons arise are shown, as well as the homeo- Graduate and Undergraduate Students domain factor code they express in wild-type mice. Lbx1 suppresses the emergence of class A neurons, and specifies the development of class B neurons (indicated). In Mathias Gierl* Lbx1LacZ/LacZ mice, class B neurons assume abnormal molecular characteristics (dI4* Martin Sieber and dI5* neurons), and express the indicated homeodomain factor combinations typical Hendrik Wildner of class A neurons (shown on the right). Katja Reuter* Dominique Bröhl* Elena Vassioutina month. We infer that signaling from the ErbB2 receptor, Malgorzata Borowiak which is enriched in T-tubules in cardiomyocytes, is crucial Michael Strehle not only during heart development but also for the correct functioning of the adult heart. Conditional ErbB2 mutants Technical Assistants provide a novel animal model of dilated cardiomyopathy and Karin Gottschling will allow for a rigorous assessment of the adverse effects of Carthrin Rudolph anti-ErbB2 antibodies on cardiac function. Andrea Leschke Tanja Schalow* Sven Buchert Selected Publications Animal Care Zechner D, Fujita Y, Hulsken J, Müller T, Walther I, Taketo Petra Krause* MM, Crenshaw EB, Birchmeier W, Birchmeier C. beta-Ca- tenin signals regulate cell growth and the balance between * part of the period reported progenitor cell expansion and differentiation in the nervous system. Dev Biol. 2003; 258:406-18.

Birchmeier, C., Birchmeier, W., Gherardi, E., Vande Woude, G. F. Met, metastasis, motility and more. Nature Reviews Molecular Cell Biology 4, 915-925 (2003)

Özcelik C, Erdmann B, Pilz B, Wettschureck N, Britsch S, Hubner N, Chien KR, Birchmeier C, Garratt AN. (2002) Con- ditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc Natl. Acad. Sci. U S A. 99(13):8880-5.

Simon Hippenmeyer, Neil A. Shneider, Carmen Birchmeier, Steven J. Burden, Thomas M. Jessell and Silvia Arber. 2002 A Role for Neuregulin1 Signaling in Muscle Spindle Differen- tiation, Neuron 36:1035-49.

Müller T, Brohmann H, Pierani A, Heppenstall PA, Lewin GR, Jessell TM, Birchmeier C. (2002) The homeodomain factor lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord. Neuron 16;34(4):551-62. 134

Molecular Control of Spinal Cord these candidate genes are initially expressed in late born, and Peripheral Nervous System postmitotic dorsal neurons. Subsequently, expression beco- Development mes restricted to distinct neuronal subpopulations within the superficial layers of dorsal horn (see figure). Neurons located within these layers are known to be involved in the processing Stefan Britsch (Helmholtz Fellow) and relaying of nociceptive and mechanosensory information. Thus, our preliminary data indicate that our candidate genes are potentially involved in the specification, terminal differentiation, and/or positioning of specific neuronal subpopulations of the superficial dorsal horn. Current work of the group focuses on the functional characterization of these genes by targeted deletion in mice.

Selected Publications

Özcelik C, Erdmann B, Pilz B, Wettschurek N, Britsch S, Hübner N, Chien KR, Birchmeier C, Garratt AN (2002) Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. PNAS 99: 8880-8885.

Our group is interested in the identification and functional Mukouyama Y, Shin D, Britsch S, Taniguchi M, Anderson DJ characterization of novel genes involved in the development (2002) Sensory nerves determine the pattern of arterial diffe- of the dorsal spinal cord and peripheral nervous system. rentiation and blood vessel branching in the skin. Cell 109: 693-705. During development, distinct cell types are generated along the dorso-ventral axis of the neural tube. Neurons that process Britsch S, Goerich DE, Riethmacher D, Peirano RI, D, and relay sensory information primarily reside in the dorsal Rossner M, Nave KA, Birchmeier C, Wegner M (2001) The portion of the spinal cord, whereas neurons that integrate and transcription factor Sox10 is a key regulator of peripheral direct motor control are located ventrally. This regionally re- glial development. Genes Dev 15: 66-78. stricted generation of different neuron types, and subsequent establishment of complex neuronal circuits, are governed by Grimm J, Sachs M, Britsch S, Di Cesare S, Schwarz-Romond extracellular signals and transcriptional networks. In order to T, Alitalio K, Birchmeier W (2001) Novel p62dok family identify novel genes involved in control of these processes, members, dok-4 and dok-5, are substrates of the c-Ret recep- we have used global gene expression analyses with high-den- tor tyrosine kinase and mediate neuronal differentiation. J sity oligonucleotide microarrays. Cell Biol 154: 345-354

We have detected several novel or only partially characterized genes with unknown functions in the development of the dorsal spinal cord. Expression analysis has revealed that many of

Spatio-temporal expression of the homeodomain transcription factor Gbx1 during embryonic development of the spinal cord. Gbx1 was identified with Affymetrix microarrays and predicted to be differentially expressed in the dorsal spinal cord. Shown are cross-sections through the spinal cord of wildtype mice at different developmental stages (E12.5 – E19.5). Gene expression is visualized by in situ hybridization. 135

Stucture of the Group

Group leader Dr. Stefan Britsch

Graduate Students Heike Brylka* Anita John*

Technical Assistants Verena Sasse

* part of the period reported 136

Genetic Analysis of Nervous Characterization of novel molecular components System Development and Function involved in pain perception

The perception of acute pain stimuli is crucial in alerting an Alistair N. Garratt (Helmholtz Fellow) organism to environmental dangers, and is thus an important survival mechanism. In contrast, chronic pain states in patients are debilitating conditions, generally refractory to current treatments, and lead to substantial losses in quality of life. Currently, considerable effort is being invested worldwide to better characterize the molecular bases of pain circuitry and enable the design of novel therapies for acute and chronic pain.

One area of particular importance for the reception of pain modalities is the superficial dorsal horn of the spinal cord, in particular, the substantia gelatinosa. This region is innervated principally by non-myelinated projections from pain-sensing peripheral neurons (nociceptors), and can be identified as a semi-translucent region in the head of the dorsal horn. Despite the significance of this area to pain processing, the molecular characteristics of the maturing and adult dorsal horn are as yet poorly defined. The development and homeostasis of multicellular organisms depends on the ability of cells to receive extracellular signals We used an Affymetrix microarray-based screen to identify and to transmit this information into the cell interior. Various genes with enriched expression in the superficial dorsal horn types of cellular response may ensue, for example cells may of the adult mouse spinal cord. These could be grouped into alter their migratory behaviour, change their proliferation various classes, including genes encoding neuropeptides, rate, progress into a final differentiated state, or undergo neuropeptide and glutamate receptors, cation channels, tran- apoptosis. We are interested in signalling systems required for scription factors, and calcium-binding proteins. Expression development and functioning of the mammalian peripheral patterns of selected genes were verified by in situ hybridiza- and central nervous systems. We employ classical knock-out tion. These include c-kit, encoding a receptor tyrosine kinase and conditional gene targeting strategies in order to address with multiple developmental functions, which is also known the roles of signalling molecules in vivo in the mouse. One to be expressed by nociceptive neurons, and the three mouse avenue of particular medical interest is the role of growth homologues of the Drosophila gene teashirt (tsh), a homeotic factor signalling in the maintenance of neuronal stem cells gene which encodes a zinc-finger protein involved in trunk and/or their progeny in the adult. identity in the fly. All four genes are widely expressed during mouse development, and have interesting expression patterns in both the peripheral and central nervous systems. Conditional mutation of the receptor tyrosine kinase ErbB2 in the nervous system

The proto-oncogenene ErbB2 (c-neu or HER2 in human) During the development of the peripheral nervous system, neurons and their axons provide signals that control development of satellite glia (orange) and Schwann cells encodes a receptor tyrosine kinase, which is frequently over- (yellow), respectively. One axonally derived signal that controls the numbers of early expressed in human tumours and is the target of an antibody- Schwann cell precursors, and which also regulates the thickness of the insulating myelin sheath made by mature Schwann cells, is provided by type III Neuregulin-1 (red arrows). based therapy for metastatic mammary carcinoma (trastuzu- This growth factor activates the ErbB2/ErbB3 receptor heteromer (black and green) mab/Herceptin). ErbB2 functions as a co-receptor for other expressed by cells of the Schwann cell lineage. Schwann cells in turn provide other signals (black arrows, circle) important for the maintenance of neurons and axons, and members of the ErbB family of receptor tyrosine kinases. In for the development of the perineurium (white ellipsoids). particular, the EGF-like growth factor Neuregulin-1 transmits signals via ErbB2/ErbB3 and ErbB2/ErbB4 heteromers. The ErbB/Neuregulin-1 signaling system is essential for normal embryonic development and continues to function in various tissues and organs in the adult animal, including the central nervous system. Previously, working together with Prof. C. Birchmeier (MDC), we employed conditional gene targeting (cre-loxP technology) to inactivate the receptor tyrosine kinase ErbB2 in myelinating Schwann cells and cardiomyocytes. We are currently focusing on analysis of mice in which ErbB2 has been mutated in the central nervous system, using a Brn4-cre transgene. These studies indicate that ErbB signaling is important for the maintenance of specific groups of neurons in the adult nervous system. 137

We are now generating knock-out and conditional alleles of the three mouse teashirt genes and have obtained a mouse strain carrying a null mutation in the c-kit gene from the Jackson laboratories. We will analyse the function of these genes in development and maintenance of the nervous system. Furthermore, as c-kit mutants can be generated as viable homozygotes, we can begin to investigate the phenotype of the pain-sensing circuitry of adult mouse mutants directly using electrophysiological techniques.

Selected Publications

Garratt, A.N., Özcelik, C., and Birchmeier, C. (2003). ErbB2 pathways in heart and neural diseases. Trends in Cardiovas- cular Medicine, 13, 80-86.

Schneider JW, Chang AY, Garratt A. (2002). Trastuzumab cardiotoxicity: Speculations regarding pathophysiology and targets for further study. Semin. Oncol, 29, 22-28.

Özcelik, C., Erdmann, B., Pilz, B., Wettschureck, N., Britsch, S., Hübner, N., Chien, K. R., Birchmeier, C. and Garratt, A.N. (2002). Conditional mutation of the ErbB2 (HER2) receptor in cardiomyocytes leads to dilated cardiomyopathy. Proc. Natl. Acad. Sci. USA, 99, 8880-8885.

Garratt, A.N., Voiculescu, O., Topilko, P., Charnay, P. and Birchmeier, C. (2000). A dual role of erbB2 in myelination and in expansion of the Schwann cell precursor pool. J. Cell. Biol., 148, 1035-1046.

Garratt, A.N., Britsch, S., and Birchmeier, C. (2000). Neure- gulin, one factor with many functions in the life of a Schwann cell. Bioessays, 22, 987-996.

Meyer, D., Yamaai, T., Garratt, A., Riethmacher-Sonnenberg, E., Kane, D., Theill, L.E. and Birchmeier, C. (1997). Isoform- specific expression and function of neuregulin. Development, 124, 3575-3586.

Structure of the Group

Group Leader Dr. Alistair Garratt

Postgraduate Students Elena Rocca Christina Schmidt

Technical Assistant Carola Griffel 138

Cellular Neurosciences neuron-glia interaction is mediated by nitric oxide (NO) which is known to be released from parallel fibres. We specu- late that these units could feedback information on a defined Helmut Kettenmann population of synapses, namely those which are enwrapped by a given microdomain.

In a collaborative grant with the Bogomoletz Institute of Physiology, Kiev, we have obtained evidence that astrocytes receive synaptic input. A transgenic animal model developed in our laboratory made it possible to identify small astrocyte compartments on the ultrastructural level.

2. How do astrocytes communicate among each other?

From experiments in cell culture and from studies in the isolated retina, it has become evident that astrocytes can communicate over large distances (<0.5 mm) via calcium signalling in the form of waves. We have found conditions to elicit and record such Ca2+ waves in slices containing the corpus callosum. In this white matter tissue the communi- The central nervous system contains two major cell popula- cation among the astrocytes is mediated by ATP release and tions, neurons and glial cells. The neurons are regarded as the activation of purinergic receptors. The calcium waves spread elements mediating the electrical activity in the brain. As a over a large distance involving more than a hundred cells. The consequence, neuroscience research of the past has focused wave travels with a low speed of about 10 m/s similar as in on this cell type. The functional role of glial cells is not as ob- culture and is thus 1,000,000 times slower than the neuronal vious: while they were first described as cells providing only action potential. In brain tissue, the calcium wave is not structural support to neurons, a series of new studies on glial restricted to astrocytes, but also activates cells of the oligo- cell function has attracted the attention of the neuroscience dendrocyte lineage and the microglial cells. Using cortex as community. It has become evident that glial cells are essential a model of grey matter, we found that neuronal activity can for the proper functioning of the brain. The different types of trigger an additional Ca2+ activity in astrocytes which spreads glial cells fulfil distinct tasks. Oligodendrocytes are the my- faster than the intrinsic astrocyte wave. This neuron-driven elin-forming cells of the central nervous system and ensure a astrocyte activity is, for instance, observed during cortical rapid signal conduction in the white matter. The role of spreading depression, a phenomenon associated with the aura astrocytes is less well defined; they provide guiding structu- of migraine. res during development and represent important elements for controlling the composition of the extracellular space mediating signals between the brain endothelium and the neuronal membrane. Microglial cells are immuno-competent cells in the brain and their functional role is best defined as the first responsive elements during pathologic events. While in the last years the group has studied aspects related to all three types of glial cells, the present research program is focused on Rat-Astrocytes (red) and microglia (green) in primary culture three topics: (1) the role of astrocytes in information process- (red: staining against GFAP ing (2) the response of microglial cells to brain injury and (3) green: ILB4-staining) the cellular properties of gliomas.

1. How do astrocytes detect neuronal activity?

In the last years, we have learned that astrocytes in cell culture have the capacity to express almost all receptors known to mediate synaptic transmission. One of our best- studied examples is the Bergmann glial cell in the cerebellum, a morphologically specialized astrocyte. We found that activity of parallel fibres, the axons of the granule cells syna- psing onto Purkinje neurons, triggers a calcium signal in Bergmann glial cells. With moderate activity, the signal can be confined to a sub region of the cell. This sub region has a morphological correlate, the microdomain. With more intense stimulation the signal spreads to the soma. This form of 139

4. What factors control activation of microglial cells?

Microglial cells are activated by any type of brain injury or pathologic disturbance. Using bacterial lipopolysaccharide (LPS) as a tool to activate cultured mouse microglia, we studied alterations in the intracellular calcium concentration 2+ ([Ca ]i) and in the receptor-evoked generation of transient calcium signals. LPS treatment led to a chronic elevation of 2+ basal [Ca ]i along with a suppression of evoked calcium signalling. Our findings suggest that chronic elevation of basal 2+ [Ca ]i attenuates receptor-triggered calcium signalling. 2+ Moreover, increased [Ca ]i is required, but by itself not sufficient, for release of NO and certain cyto/chemokines. Eleva- 2+ tion of basal [Ca ]i could thus prove to be a central element in the regulation of executive functions in activated microglia.

The figure displays the extent of biocytin coupling of a single astrocyte in cortex. Note that astrocyte endfeet delineate a blood vessel. 5. Are microglial cells important for neuronal reorganization after injury?

A candidate for signalling neuronal injury to microglial cells is the chemokine CCL21, since damaged neurons express CCL21. Investigating microglia in acute slices and in culture, we demonstrate that CCL21 triggers a Cl- conductance increase. Moreover, CCL21 triggers a chemotactic response, which is sensitive to Cl- channel blockers. Both types of responses are mediated by activation of CXCR3 and not CCR7 receptors indicating that in brain, CCL21 acts via a different receptor system than in lymphoid organs. We have now tested the impact of CXCR3 signalling on cellular responses after entorhinal cortex lesion. In wild type mice, microglia migrate within the first 3 days after lesion into the zone of axonal degeneration, where 8 days after lesion denervated dendrites of interneurons are subsequently lost. In contrast, the recruitment In an experimental tumor model in mouse, glioma cells (labelled red) attract neural precursor cells (labelled green) of microglia was impaired in CXCR3 knockout mice and, strikingly, denervated distal dendrites were maintained in zones of axonal degeneration. No differences between wild type and knockout mice were observed following facial nerve axotomy, as a lesion model for assessing microglial proliferation. This shows that CXCR3 signalling is crucial in microglia 3. What are the physiological features of microglial recruitment, but not in proliferation, and this recruitment is an cells in brain tissue? essential element for neuronal reorganization. This research is funded by a binational grant with Erik Boddeke, Groningen. Microglial cells are the major immunocompetent cells in the brain and express many features of monocytes. This includes signalling cascades well described in the immune system 6. What are the physiological properties of gliomas involving chemokines and cytokines and their receptor and how do they compare to normal glia? systems. In this project, we addressed the question whether microglia would also express receptors to sense neuronal The majority of tumors of the central nervous system are activity. We have recently developed an in situ model which thought to originate from glial cells. These include astrocyto- allows to study the physiological responses of resting and mas, oligodendrogliomas, and the most malignant (and untrea- activated microglia. This enables us to characterize the func- table) brain tumor, the glioblastoma multiforme. We study the tional receptors and the physiological phenotype of microglia cellular properties of these tumor cells and compare them to in situ. Using this approach, we could identify microglial normal glial cells with respect to their physiological properties receptors for GABA, the major inhibitory transmitter of the and their abilities to proliferate and migrate. Currently, we ad- CNS. Activation of the GABAB receptors suppressed indi- dress the question whether microglial cells influence tumor cators of microglial activation such as the release of Il-6. A cell behaviour. In a long-term slice culture, we injected a defi- similar reduction in proinflammatory mediators was found ned amount of tumor cells and quantified their migration with- with activation of purinergic receptors which are important in tissue. We found that microglial cell depletion from the slice signalling molecules for astrocyte activity. These findings slowed tumor invasion. Thus, the presence of microglial cells support the hypothesis that microglial cells are less prone to promotes the invasion of tumor cells. This research is funded activation when they sense normal neural activity. by a binational grant with Bozena Kaminska, Warsaw. 140

Selected Publications

Kuhn S. A., van Landeghem F. K. H., Zacharias R., Rappert A., Pavlovic S., Hoffmann A., Nolte C., Kettenmann H.,

(2004) Microglia express GABAB receptors to modulate nitric oxide and interleukin-6 release, Mol. Cell. Neurosci. 25:312-322.

Peters O., Schipke C.G., Hashimoto Y., Kettenmann H. (2003) Different mechanisms promote astrocyte Ca2+-waves and spreading depression in the mouse neocortex, J. Neu- rosci., 23:9888-9896 .

Hoffmann, A., Kann, O., Ohlemeyer, C., Hanisch, U-K., and Kettenmann, H. (2003) Elevation of basal intracellular calcium as a central element in the activation of brain macrophages (microglia):suppression of receptor-evoked calcium signalling and control of release function, J. Neurosci. 23:4410-4419.

Makara J.K. , Rappert A., Matthias K., Steinhäuser C., Spät A. and Kettenmann H. (2003) Astrocytes from mouse brain slices express ClC-2 mediated Cl- currents regulated during development and after injury. Mol. Cell Neurosci. 23:521- 530.

Rappert A., Biber K., Nolte C., Lipp M., Schubel A., Bao L., Gerard N. P., Gerard C., Boddeke H. W. G. M. and Ketten- mann H. (2002) Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl current and chemotaxis in murine microglia. J. Immunol. 168:3221-3226.

Structure of the Group

Group Leader Technical Assistants Prof. Dr. Helmut Kettenmann Bärbel Girresch Christiane Gras Assistant to the Group Leader Brigitte Gerlach (part time) Meino Gibson Irene Haupt Michaela Seeger (part time) Scientists Horst Kagelmaker Dr. Rainer Glass Dr. Katrin Faerber Secretariat Dr. Anja Hoffmann Birgit Jarchow Dr. Christiane Nolte (part time) Carola Schipke Dr. Michael Synowitz

Graduate Students Brigitte Haas Antje Heidemann Hannes Kiesewetter Darko Markovic Ulrike Pannasch Andreas Phillips Liping Wang 141

Brain Energy Metabolism lopmental manner. In previous studies, we found that the catalytic activity of COX is regulated according to the energy level of the cell: binding of ATP, the indirect product of the Susanne Arnold (Emmy Noether Research Group) COX reaction, to subunit IV of COX at high ATP/ADP ratios leads to an allosteric inhibition of the enzyme. For the first time, we could show that, in astrocytes from rat brain, the transcription of COX subunit IV isoforms is regulated by the availability of oxygen, the substrate of the enzyme. In astrocytes, the lack of oxygen leads to a switch in the expression pattern from subunit IV-1 isoform under normoxic conditions to subunit IV-2 isoform under hypoxia. The functional consequences of the COX isoform expression on astrocytic energy metabolism under hypoxic conditions will be studied in more detail.

Selected Publications

Kadenbach B., Arnold S., Lee I., Hüttemann M. (2004).The possible role of cytochrome c oxidase in stress-induced apoptosis and degenerative diseases. Biochim. Biophys. Acta in press. Our group investigates astrocytes, the most prominent cell group in the mammalian brain, and the regulation of their Ludwig B., Bender E., Arnold S., Hüttemann M., Lee I., energy metabolism in coordination with neuronal activity Kadenbach B. (2001).Cytochrome c oxidase and the regu- under physiological and pathological conditions. Astrocytes, lation of oxidative phosphorylation.Chembiochem Eur. J. strategically well positioned between capillaries and neurons, Chem. Biol. 2, 392-403. can regulate energy substrate availability, such as glucose, and energy production in response to neuronal energy consumption. Structure of the Group

We apply various fluorophores and protein sensor molecules to Group Leader register changes in cytosolic ion and second messenger con- Dr. Susanne Arnold centrations in astrocytes upon neuronal activation, correlating them with changes of parameters of the energy metabolism, Graduate Students such as mitochondrial membrane potential, NAD(P)H content, Susann Härtig and mitochondrial calcium. So far, we could show that as a Volker Horvat consequence of increases in astrocytic cytosolic calcium, the mitochondrial membrane potential transiently depolarizes and Technical Assistant the NAD(P)H content decreases. The underlying mechanisms Angelika Mai are not well understood yet and will be studied in detail.

Glucose is the major energy substrate in the brain, which is A) B) Two photon microscope image of rhodamine 123 stained mitochondria from mainly taken up by astrocytes and converted into lactate to mouse astrocytes. C) Structure of the dimeric cytochrome c oxidase complex from bovine heart [Tsukihara fulfill neuronal energy requirements. Taking into account the et al. (1996) Science 272, 1136] importance of glucose for the energetic balance of brain cells, Coordinates (PDB entry locc) were processed with Raswin 2.6. we studied the impact of hypoglycemia on astrocytic calcium levels and signaling. We found that hypoglycemia induces an A increase in cytosolic calcium and an impairment of neurotransmitter-triggered calcium signaling in astrocytes. The hormone, 17-estradiol, protects astrocytes from the impact of hypoglycemia on resting Ca2+ levels and on neurotransmitter- B triggered Ca2+ signaling. The mechanism of the hormone action is a focus of our further studies. C Cytochrome c oxidase (COX), the terminal and rate-limiting enzyme of the mitochondrial respiratory chain, is engaged in oxidative energy metabolism. The mammalian enzyme (see figure) is composed of 3 catalytic, mitochondrial-encoded and 10 regulatory, nuclear-encoded subunits. In mammals, isoforms have been identified for subunits IV, VIa, VIb, VIIa and VIII, which are expressed in a tissue specific and deve- 142

Developmental Neurobiology signalling, characterization of specific axonal Ig superfamily members, and induction of actin-rich microprocesses along axons. Since activity-dependent processes are less under- Fritz G. Rathjen stood, we became also interested in identifying cell surface proteins on neurons which are modulated by neuronal activity.

Tenascin-R induces microprocesses along neurite shafts

During development, axons and dendrites generate finger-like protrusions called filopodia or microprocesses along their shafts. Several observations have led to the proposal that these filopodia most likely initiate synapse formation by rea- ching out to neighbouring neurites and, therefore, might play an inductive role in the generation of synapses. In a search for extracellular signals that affect the formation of microprocesses in tectal neurons, we identified Tenascin-R (TN-R), a glycoprotein of the extracellular matrix. The formation of microprocesses by TN-R extending laterally along the neuri- tic shaft was time and dose-dependent and was accompanied Activity–independent and –dependent processes by a rearrangement of the cytoskeleton. Contactin (F11), a regulate the wiring of the nervous system cell adhesion molecule of the Ig superfamily that is associated with the plasma membrane via a lipid anchor, was found to The establishment of precise and selective synaptic connec- mediate the effects of TN-R. These observations suggest that tions between neurons during embryonic and early postnatal TN-R might induce a transition from long distance growth of development is essential for the proper functioning of the ner- tectal interneurons to differentiation, including the formation vous system. A longstanding goal of many neuroscientists is of microprocesses. Current studies focus on the intracellular to understand the formation of these neuronal connections signal transduction cascade activated by TN-R via contactin throughout the nervous system which most likely involves a to induce filopodia along neurite shafts. complex series of events. However, in principle, two overlapping mechanisms might be distinguished: processes that are independent of electric activity and processes that are depen- cGMP-mediated signalling in sensory axon dent on electric activity of the neurons. Neuroscientists pathfinding within the spinal cord assume that activity-independent mechanisms establish the basic pattern of connectivity that becomes subsequently The signal transduction machinery within the growth cone refined by activity-dependent processes. can be modulated by the levels of the cyclic nucleotides cAMP and cGMP. Our studies on the pathfinding of sensory Activity-independent molecular processes have been analy- axons indicated that the cGMP-dependent protein kinase I zed in detail in past years and several molecular guidance (cGKI) is required for sensory axons to find their way within cues in the environment of axons, as well as guidance recep- the dorsal root entry zone (DREZ) of the spinal cord. Once tors on the surface of growth cones, have been characterized. sensory axons arrive at the DREZ of the spinal cord, each For a general overview, the activity-independent axonal gui- axon bifurcates into a rostral and caudal branch extending dance factors have been categorized as being attractive or over several segments. After a waiting period, collaterals repulsive to growth cones. These factors belong also to diffe- grow out from these longitudinal stem axons and form rent protein families including neural members of the Ig lamina-specific projections within the grey matter. In the superfamily, semaphorins, netrins, ephrins, neuropilins, absence of cGKI, we observed, by DiI tracing and antibody plexins, Eph-kinases and several extracellular matrix staining, that many sensory axons fail to bifurcate correctly at proteins. Our previous work focussed primarily on several Ig the DREZ and instead grow directly to the central canal. superfamily members such as F11, L1, neurofascin, NrCAM These axon guidance defects in cGKI-deficient mice result in and neurotractin. a substantial reduction of the amplitude of the nociceptive flexion reflex. Our studies, therefore, demonstrated that In contrast to the activity-independent mechanisms, the speci- cGMP signalling via cGKI is important for directing axonal fic manner in which electric activity influences the refine- growth of sensory axons. ment of synapses is still a matter of speculation and is controversial even in relatively well-established systems such as the To further our understanding of these observations, we are visual system. currently investigating up- and downstream components of the cGKI-signalling pathway within sensory axons. We are In the past granting period, the research activities of our using biochemical approaches, in vitro outgrowth studies, as group focussed on different aspects of axonal pathfinding re- well as genetic mouse models to unravel components of the gulated by activity-independent processes including cGMP cGMP signalling cascade within sensory axons. This includes 143

Selected Publications

Erdmann, B., Kirsch, F.P., Rathjen, F.G., Moré, M.I. (2003) N-cadherin is essential for retinal lamination in the zebrafish. Dev. Dyn., 226, 570-577.

Alberts, P., Rudge, R., Hinners, I., Muzerelle, A., Martinez- Arca, S., Irinopoulou, T., Marthiens, V., Tooze, S., Rathjen, F., Gaspar, P., and Galli, T. (2003) Cross-talk between tetanus neurotoxin-insensitive vesicle-associated membrane protein- mediated transport and L1-mediated adhesion. Mol. Biol. Cell., 14, 4207-4220.

Schmidt, H., Werner, M., Heppenstall, P.A., Henning, M., Moré, M.I., Kühbandner, S. , Lewin, G.R., Hofmann, F., Feil, Induction of filopodia on neurites by the extracellular matrix glycoprotein Tenascin-R (TN-R). These highly dynamic microprocesses might be important for initial contacts to R., and Rathjen, F.G. (2002) cGMP-mediated signalling via neighbouring neurites to establish synapses. cGKIa is required for the guidance and connectivity of sensory axons. J. Cell Biol., 159, 489-498.

the characterization of guidance signals acting within the Bixby, J.L., Baerwald, K.D., Wang, C., Rathjen, F.G. and DREZ as well as their corresponding guidance receptor(s) on Rüegg, M.A. (2002) A neuronal inhibitory domain in the N- sensory axons whose activation elevates intracellular cGMP terminal half of agrin, J. Neurobiol., 50, 164-179. levels and, subsequently, triggers cGKI. The identification of the soluble or particulate guanylyl cyclases, which are invol- Zacharias, U., Leuschner, R., Nörenberg, U., and Rathjen, ved in the generation of cGMP and the phosphodiesterase(s) F.G. (2002) Tenascin-R induces actin-rich microprocesses which degrade cGMP, are also of primary interest. The analy- and branches along neurite shafts. MCN, 21, 626-633. sis of downstream targets which are phosphorylated by cGKI should also help to define the function of cGKI in sensory growth cone steering further. Finally, we will apply our kno- Structure of the Group wledge about cGMP signalling in sensory axons to other relevant neuronal cell populations in the central nervous system Group Leader that express cGKI during development. Prof. Dr. Fritz G. Rathjen

Scientists The proteolytical processing of CALEB is facilitated Dr. René Jüttner by electric activity and in the absence of CALEB Dr. Michael Koroll synapse formation is impaired Dr. Margret Moré* Dr. Hannes Schmidt As described above, the formation of precise synaptic Dr. Ute Zacharias connections in the brain is critically dependent on electric activity, which is important for the elimination of inappropriate Graduate and Undergraduate Students connections and for the fine-tuning and stabilization of ap- Aleksei Babich propriate ones. The molecular constituents mediating these Debashish Das processes are largely unknown. In an attempt to characterize Christopher Patzke components by which electric activity influences the develop- Eva Rodriquez Aznar ment of synapses, we have searched for cell surface proteins Michael Schäfer modulated by neural activity. To date, we have identified two Susanne Schäffer proteins, termed CALEB and CAR, modulated on the surface Thomas Unsöld of neurons by activity-dependent processes. Further studies have revealed that neuronal activity facilitates the proteoly- Technical Assistants tical processing of the transmembrane protein CALEB result- Hannelore Drechsler ing in a membrane associated form with an exposed EGF Madlen Driessner domain. In order to study the role of CALEB in synapse for- Mechthild Henning mation, a CALEB-deficient mouse was generated. The analysis of acute slices of the colliculus superior from these mice Secretariat revealed synapses functionally distinct from wild-type synap- Birgit Cloos (part time) ses at early but not at mature stages. CALEB-deficient synapses revealed a reduction of the frequency of spontaneous * part of the period reported EPSCs and IPSCs which are due to deficits within the pre- synapse. These findings indicate that CALEB signalling is required for the molecular development of synapses in early stages. 144

Growth Factors and Regeneration orthologs of mec-2 are essential for normal mechanotransduction.

Gary Lewin Mining the genome for sensory neuron markers Sensory neurons in the dorsal root ganglia can be classified neurochemically or morphologically. However, probably the most important characteristic of sensory neurons is the modality of peripheral stimulus that they preferentially detect. Thus some neurons respond to intense mechanical stimuli (nociceptors) and others respond only to the movement of the skin (Rapidly adapting mechanoreceptors). We have recently started to try and identify specific markers of these different physiological types by using genome wide microarray screens. We have taken advantage of mice with targeted deletions of neurotrophic factor genes that lose specific types of physiologically defined mechanoreceptors. For example, in mice null for the neurotrophic factor NT-4, one type of rapidly adapting mechanoreceptor so called D-hair receptors, are lost because they require specifically NT-4 for trophic support in the adult animal (Stucky et al. 2002a). We took advantage of this phenomenon to screen, using oligonucleotide microarrays, for genes that might only be expressed in Sensory neurons of the dorsal root ganglia allow us to detect D-hair receptors. One such gene found in this screen was a stimuli to the body surface that lead directly to the sensations T type calcium channel that functions to enhance the mecha- such as touch and pain. In my group, we are interested in the nical sensitivity of this receptor type (Shin et al. 2003). We genes that allow these neurons to transduce different types of are extending this approach at the moment to find functio- stimuli. In addition, we also study the genetic programs con- nally important marker genes of other mechanoreceptor types trolled by growth factors that specify sensory neuron function including nociceptors. or their connections with spinal cord circuits. Transduction in a dish In addition to examining the function of candidate genes in Molecular Basis of Mechanotransduction sensory transduction the laboratory is also using electrophysiological techniques to characterize sensory transduction in Role of mec genes single cells. For example, we use cultured sensory neurons to Mechanotransduction is the process whereby receptor pro- characterize with whole cell patch clamp techniques the ionic teins present in the endings of sensory neurons are able to currents underyling thermal stimuli (Stucky et al. 2002b). detect mechanical stimulation of the tissue they innervate. We Recently, we have established methodologies to measure have used information from genetic experiments with the nematode worm C.elegans to identify possible vertebrate candidate proteins that might detect mechanical stimuli. Genetic screens for touch insensitive worms have turned up around 15 Different mechanoreceptor types in the dorsal root ganglion. In the middle is a schema- genes whose function is necessary to confer touch sensitivity. tic drawing of the dorsal root ganglia that contains the cells bodies of the sensory neurons. Neurons are colour coded according to their sensory modality. For each These genes were named Mec for mechanically insensitive neuronal type a typical recoding of the response to a ramp and hold indentation of the and we have focused on identifying a role mammalian ortho- skin is shown. The responses of neuronal types that detect small non-painful stimuli (touch receptors) are coloured blue and those detecting intense noxious stimuli are logs of these genes in mammalian touch sensation. Some of shown in red (pain receptors). these genes encoded membrane ion channels that were proposed to open upon movement or displacement of the plasma membrane. We have recently shown that a mouse protein (BNC1/ASIC2) with significant homology to the worm ion channels is required for mice to properly discriminate touch stimuli (Price et al. 2000). Other work in the lab has concentrated on establishing whether the BNC1/ASIC2 ion channel works in concert with other ion channel subunits (eg. the DRASIC protein) to detect mechanical forces (Price et al. 2001). The mec genes in C.elegans have been proposed to work together in a mechanotransduction complex. Another component of this complex is the membrane protein Mec-2 that forms a hairpin in the membrane and might regulate the activity of the mechanotransducing channel. We have cloned new vertebrates homologues of this gene and have created mouse knockout models to characterize the in vivo function of these genes. Our data indicate that also the mammalian 145

mechanically-gated ionic conductances in single sensory Stucky C.L., Rossi J., Airaksinen M.S., Lewin G.R. (2002b) neurons and we are investigating the effect of extracellular GFRa2/neurturin signalling regulates noxious heat transduc- matrix factors on this these channels. tion in IB4-binding mouse sensory neurons. J Physiol. 545, 43-50. Hearing and touch Hereditary deafness is a relatively common phenomenon and Heppenstall, P.A. and Lewin, G.R. (2001). BDNF but not a large number of genes have been identified that when muta- NT-4 is required for normal flexion reflex plasticity and ted lead to deafness in mouse and humans. Recently we have function. Proc. Natl. Acad. Sci. USA 98, 8107-8112. started working with several deaf mutant mice to examine whether genes required for normal mechanotransduction in the inner ear may also be required for normal cutaneous Structure of the Group sensation. Our data indicate that members of the unconven- tional myosin protein family have a common function in Group Leader sensory neurons and in hair cells, mechanotransducing cells Prof. Dr. Gary R. Lewin of the inner ear. In both cell types, these proteins may function to regulate the adaptation of the mechanotransduction Scientists channels. We are currently working on further hearing genes Dr. Paul Heppenstall* that may also affect cutaneous mechanosensation. The same Dr. Andreas Eilers genes that we study in the mouse are also mutated in humans Dr. Jing Hu and it is possible that the perception of cutaneous touch Dr. Carlos Martinez-Salgado* stimuli is altered in such patients. Dr. Parvinder Rathee Dr. Regina Bönsch

Regulation of sensory synaptic connections in the Graduate Students spinal cord Jung-Bum Shin* Christiane Wetzel The synaptic connections made by sensory neurons in the Gireesh Anirudhan spinal cord underlie reflexes evoked by innocuous or noxious Alexandra Seifert stimuli. We have recently developed an in vitro electrophy- Nevena Milenkovic siological preparation to study such reflexes in the mouse Rabih Moshourab (Pesquero et al., 2000). This technique, together with the use Jochen Decker (diplom student)* of knockout mice, recently allowed us to identify the neuro- Claudius Müller (diplom student)* trophin, Brain derived neurotrophic factor (BDNF) as a functionally important pain neuromodulator released by sensory Technical Assistants neurons onto the spinal cord neurons (Heppenstall and Lewin Anke Kanehl 2001). In collaboration with other MDC groups, we have Heike Thränhardt developed this technique to provide high quality phenotype Anja Wegner information on many different mouse mutants to identify genes regulating the construction or function of spinal refle- * part of the period reported xes (Müller et al. 2002; Schmidt et al. 2002). This data will provide insights into the genes needed to construct the somatosensory system and possibly reveal new drug targets for the treatment of acute and chronic pain.

Selected Publications

Shin J.-B., Martinez-Salgado, C., Heppenstall P.A., Lewin G.R., (2003) A T -type calcium channel required for the function of a mammalian mechanoreceptor. Nature Neurosci. 6:724-30.

Müller, T., Brohmann, H., Pierani, A., Heppenstall, P.A., Lewin, G.R., Jessell, T.M., and Birchmeier, C. (2002). The homeodomain factor Lbx1 distinguishes two major programs of neuronal differentiation in the dorsal spinal cord. Neuron 34, 551-562.

Stucky C.L., Shin J.-B., Lewin G.R. (2002a) Neurotrophin-4: a survival factor for adult sensory neurons. Current Biology 12, 1401-1404. 146

Proteomics and Molecular interacting protein, GIT1, was discovered. In HD patient Mechanisms of Neurodegenerative brains, GIT1 is present in neuronal inclusions, suggesting that Disorders it is involved in disease pathogenesis.

Erich Wanker Systematic approach: Large scale mating two-hybrid screens

Gene and protein function have become the major areas of inquiry on the way to a full description of all biological processes and their malfunction. Proteins rarely act alone in the cell. Their interaction patterns provide valuable information about their function. We are currently generating a comprehensive human protein-protein interaction network using an automated yeast two-hybrid (Y2H) system. This large-scale, systematic study is based on interaction mating in a 384-well matrix format. Two different haploid yeast strains of opposite mating type (MAT and MATa) are mated after transformation with plasmids encoding activation domain and DNA- binding domain fusion proteins, respectively. Interactions are detected via transcriptional reporter activation. A non-redundant set of more than 3,600 cDNAs has been subcloned into The sequencing of the human genome has provided the basis DNA-binding and activation domain vectors. Additionally, for the systematic analysis of protein function. The main 2,000 human full length ORFs were cloned into two-hybrid objective of our work is to generate knowledge about human vectors. To handle the large number of yeast clones, an auto- proteins and to link them to disease processes using high- mated version of the Y2H system was set up, using pipetting, throughput functional proteomics technologies. Through the spotting, and gridding robots. A screening of about 25 million large-scale identification of physical protein-protein interac- potential interactions was performed and allowed the initial tions (PPIs) by automated yeast two-hybrid screening, we are identification of about 2,000 PPIs. These interactions are creating comprehensive protein interaction maps in order to currently validated and verified using bioinformatics, cell characterise proteins and to understand the regulatory proces- biology, and biochemical methods with special attention to ses that control the biology of living organisms. These studies disease proteins. are also intended to identify new targets for therapeutic intervention. Closely linked to these goals is our work on the function and dysfunction of proteins involved in late onset High-throughput protein expression and production neurodegenerative diseases like Huntington’s (HD), Parkin- of protein arrays son’s (PD) and Alzheimer’s (AD) disease. These disorders are characterised by the accumulation of intra- and extracellular Recombinant human proteins are valuable resources for many protein aggregates, considered critical for disease pathogene- applications in functional genomics and proteomics. We have sis. We have developed cell-based and in vitro drug screening developed an approach that allows the parallel expression of assays for the identification of small molecules that delay ~13,500 His-tagged fusion proteins from a human brain aggregate formation. Lead compounds have been identified cDNA library in 384-well microtitre plates. Crude protein and are currently tested for their activity in different trans- extracts are high-density gridded onto 22 x 22 cm membrane genic in vivo model systems. filters with a spotting robot. We then probed the arrays with an anti-RGS-His antibody and identified about 2,300 recombinant human proteins available for further interaction Generation of a protein-protein interaction network studies. for Huntington’s disease by automated two-hybrid screening The high-density spotted membranes were probed with bacterial protein extracts containing overexpressed recombinant In the past two years, we have developed a strategy combi- human GST fusion proteins as baits. Reproducible interaction ning library and matrix yeast two-hybrid screens to create a maps were obtained for 15 bait proteins involved in neuro- highly connected protein-protein interaction network for degenerative diseases, endocytosis, and gene expression. In Huntington’s disease (HD). We used 40 proteins with 10 total, the interaction studies with high-density spotted protein different huntingtin (htt) fragments for library screens and arrays allowed the identification of about 150 PPIs, which are tested all identified proteins for interactions with all bait pro- currently validated by means of pull-down assays, mass teins in a systematic array-mating screen. In total, a network spectrometry, co-localisation experiments and functional consisting of 86 proteins involved in 188 protein-protein assays. interactions was generated, 16 of which were confirmed by in vitro binding assays, co-immunoprecipitations, or co-localisation studies. Sixteen uncharacterised proteins could be functionally annotated and a G protein-coupled receptor kinase 147

necessary and sufficient for the binding to p97. It is localised downstream of the polyQ tract and contains two putative ubiquitin interacting motifs (UIMs). The p97 N-terminal domain (aa 1-200) was crucial for the ataxin-3 interaction.

Testing whether p97 modulates ataxin-3 aggregation, we found that increasing concentrations of p97 up to equimo- larity stimulated it, whereas a 3 to 4-fold molar excess of p97 strongly suppressed it. This was confirmed in a cellular model. In a transgenic Drosophila model of SCA3, both overexpression of human p97 and heterozygous loss of the protein suppressed polyglutamine induced neurodegeneration. More- over, neuronal intranuclear inclusions containing aggregated ataxin-3 showed a significant size reduction. Alteration of p97 levels in neurons could open up new avenues for therapeutic intervention in SCA3.

Characterisation of amyloid aggregation inhibitors using in vitro and in vivo model systems

Immunofluorescence microscopy image of COS1 cells expressing GIT1 and huntingtin. GIT1 was identified by automated yeast two-hybrid screening using huntingtin as bait. It Neuronal protein aggregates are a hallmark of neurodegene- binds to huntingtin and stimulates the accumulation of membrane vesicles in early rative disorders, like Alzheimer’s (AD), Parkinson’s (PD), endosomes. Moreover, GIT1 promotes the accumulation of huntingtin aggregates in mammalian cells. and Huntington’s disease (HD). Animal studies suggest that removal of aggregates ameliorates the disease course. Especi- ally for HD, it is now widely assumed that aggregation inhibition in patients will slow down disease progression.

Co-aggregation of mutant and wild-type huntingtin To search for polyQ aggregation inhibiting compounds, we performed a screen using an automated filter retardation Huntington’s disease (HD) is a progressive neurodegenera- assay. We detected around 300 compounds, a major group tive disorder caused by an expanded polyQ repeat in the being benzothiazoles, significantly reducing htt aggregate N-terminal part of the htt protein. The elongated protein self- formation in a dose-dependent manner. Some were then found assembles into amyloid-like aggregates, while wild-type htt to inhibit polyQ aggregation in mammalian cells. Currently, does not aggregate. Using cell-free and cell-based assays, we cytotoxicity assays are conducted. The most promising com- found that mutant htt promotes the aggregation of wild-type pounds will then be studied in transgenic mouse models of htt by a nucleation-dependent process, causing the formation HD. The HD R6/2 transgenic mouse model was established of SDS-resistant co-aggregates with a fibrillar morphology. for systematic drug screens. The mice are transgenic for exon Conversely, mutant htt does not promote the fibrillogenesis of 1 of the human HD gene with a greatly expanded CAG repeat the polyQ-containing protein NOCT3 or the polyQ-binding and recapitulate many of the features of the human disease. At protein PQBP1, although they are recruited into inclusions the moment, the benzothiazole derivatives PGL-135 and containing mutant htt aggregates in mammalian cells. Htt PGL-137 are studied. The effects are assessed in behavioural fibril formation is a highly selective process, depending on assays like motor ability and grip strength tests. Brains are polyQ tract length and on other determinants in the htt amino examined immunohistologically and htt aggregates are quan- acid sequence. Our data suggest that mutant and wild-type htt tified using a filter retardation assay. Compounds able to also co-aggregate in neurons of HD patients and that the reduce aggregate formation and neurological symptoms in resulting loss of wild-type htt function may contribute to these mice will be selected as candidates for clinical trials. pathogenesis.

Selected Publications P97 selectively mitigates ataxin-3 aggregation and toxicity in Drosophila Busch, A., Engemann, S., Lurz, R., Okazawa, H., Lehrach, H. and Wanker, E. (2003) Mutant Huntingtin Promotes the Spinocerebellar ataxia type 3 (SCA3) is an autosomal, domi- Fibrillogenesis of Wild-type Huntingtin. J Biol Chem 278 nantly inherited neurodegenerative disorder caused by an (42), 41452-41461. expanded polyQ sequence in the ataxin-3 protein. Function of the protein and the molecular mechanism of SCA3 are un- Laurine, E., Grégoire C., Fändrich M., Engemann, S., known. We have isolated p97, a AAA ATPase, copurifying Marchal, S., Thion, L., Mohr, M., Monsarrat, B., Michel, B., with ataxin-3. Protein p97 selectively binds to ataxin-3 but Dobson, CR., Wanker, E., Èrard, M., and Verdier, MM. not to other polyQ-containing proteins such as htt, indicating (2003) Lithostathine quadruple helical filaments form pro- that it is not a polyQ-binding protein. In a series of experi- teinase K resistant deposits in Creutzfeldt-Jakob disease. ments, we found that a 70 amino acid ataxin-3 fragment was J Biol Chem 278(51), 51770-51778. 148

Harjes, P. and Wanker, EE. (2003) The hunt for huntingtin function: interaction partners tell many different stories. TiBS 28(8), 425-433.

Perutz, MF., Pope, BJ., Owen, D., Wanker, EE. and Scherzin- ger, E. (2002) Aggregation of proteins with expanded glutamine and alanine repeats, of the glutamine-rich and aspara- gine-rich domains of Sup35, and of the amyloid ß-peptide of amyloid plaques. Proc Natl Acad Sci USA 99(8), 5596-5600.

Heiser, V., Engemann S., Bröcker W., Dunkel I., Boeddrich A., Waelter S., Nordhoff E., Lurz R., Schugardt N., Rauten- berg S., Herhaus C., Barnickel G., Böttcher H., Lehrach H., Wanker E.E. (2002). Identification of benzothiazoles as potential polyglutamine aggregation inhibitors of Hunting- ton’s disease by using an automated filter retardation assay. Proc Natl Acad Sci USA 99(4), 16400-16406.

Structure of the Group

Group Leader Prof. Dr. Erich Wanker

Scientists Technical Assistants Dr. Annett Böddrich Claudia Abraham Dr. Anja Dröge Maik Faltysek Dr. Klaus Genser Anja Fritzsche Heike Göhler Gerlinde Grelle Christian Hänig Stephanie Haase Dr. Phoebe Harjes Anna Happe-Kramer Dr. Martin Herbst Tina Kausel Susanne Kostka Susanne Köppen Dr. Maciej Lalowski Sascha Mintzlaff Dr. Eva-Christina Müller Susanne Rautenberg Dr. Albrecht Otto Anke Schönherr Dr. Jaana Suopanki Margitta Schümann Dr. Ulrich Stelzl Nancy Schugardt Dr. Martin Strödicke Martina Zenkner Dr. Stephanie Wälter Uwe Worm Secretariat Erika Pisch Graduate and Undergraduate Students Branka Cajavec Dagmar Litscher Engin Toksöz 149

Neurodegeneration phila, and the rat. Dynamin-related GTPases are involved in fusion and fission processes of membranes everywhere in eucaryotic cells, f.e. vesicle formation at the plasma mem- Christiane Alexander brane, the endosome, or at the golgi apparatus. A novel aspect of recently identified members of the Dynamin protein family, like OPA1, DNM1 and Mitofusins, is their involvement in the fusion and fragmentation of mitochondria. Defects in these proteins lead to an abnormal distribution of mitochondria in the cell, as well as to deficits in mitochondrial function, f.e. respiration, or apoptosis. First ideas on the function of Dnm1 and Mitofusins have been developed, whereas for OPA1 elucidation of its role in the cell and in the nervous system remains to be established. We are gaining first insights into OPA1 function by the identification of interaction partners via yeast-two-hybrid screens, the structural characterization of OPA1 protein domains, and the generation of animal models.

Visualization of mitochondria. Immunostaining of HeLa cells with anti-cytochrome C antibody (green) and simulta- Autosomal dominant optic atrophy (adOA) is the most preva- neous live staining of the same cells with Mitotracker (red). lent hereditary optic neuropathy in humans resulting in a progressive loss of visual acuity, centrocoecal scotoma, and bila- teral temporal atrophy of the optic nerve with an onset within the first two decades of life. adOA occurs with an estimated disease prevalence of between 1:12,000 (Denmark) and 1:50,000. The disease is highly variable in expression and shows incomplete penetrance in some families. Histopatholo- gical post-mortem examination of donor eyes suggests that the fundamental pathology of adOA is a primary degeneration of retinal ganglion cells followed by ascending atrophy of the optic nerve.

OPA1 – the gene causing autosomal dominant optic atrophy

The predominant locus for this disorder (OPA1, OMIM #165500) was mapped by linkage analysis in large Danish pedigrees to a 1.4 cM interval on chromosome 3q28-q29 flanked by markers D3S3669 and D3S3562. By positional cloning, we were able to identify the underlying disease gene, OPA1, which spans about 100 kb of genomic sequence and is divided into 31 exons. The human OPA1 (hOPA1) cDNA was first described with a length of 5864 bp containing an open reading frame (ORF) of 2883 bp. Most mutations identified in adOA patients reside in the GTPase domain and in the exons coding for the very C-terminus of the OPA1 protein. A splicing hot-spot involving exons 4, 4b, 5 and 5b was discovered recently, without evidence for mutations being identified in these exons in any OPA1 patients screened, so far.

The OPA1 GTPase is a mitochondrial protein

The OPA1 protein consists of a classical N-terminal mitochondrial import signal, a coiled-coil domain (CC1), a GTP- ase, a middle domain of unknown function, and a C-terminal coiled-coil domain (CC2). OPA1 shows homology to dynamin-related large GTPases from salmon, C.elegans, Droso- 150

Expression analysis by Northern blot hybridisations revealed Alexander, C, Votruba, M, Pesch UEA, Thiselton, DL, Mayer, that OPA1 was ubiquitously present in all tissues examined S, Moore, A, Rodriguez, M, Kellner, U, Leo-Kottler, B, with the highest transcript level observed in retina, followed Auburger, G, Bhattacharya, SS, Wissinger, B. (2000). OPA1, by brain, testis, heart and skeletal muscle. Preliminary data encoding a dynamin-related GTPase, is mutated in autosomal from in-situ hybridization (ISH) experiments indicate predominant optic atrophy linked to chromosome 3q28. Nature dominant expression of the OPA1 gene in the ganglion cell Genet. 26, 211-215. layer (GCL) which is consistent with the hypothesis of the pathophysiology of ADOA. Structure of the Group

Analysis of a Drosophila OPA1-model Group Leader Dr. Christiane Alexander In Drosophila, the genetic regulation of the visual system has been successfully studied in the past, proving that the fly is an Graduate Students excellent model for ocular diseases affecting photoreceptor Maja Fiket cells in humans. The OPA1 gene is phylogenetically highly Dr. Vasudheva Reddy Akepati conserved and the organization of the homologous gene in Marco DelBarba Drosophila is less complex than in humans. In the fly, it spans Anita Bulczak 4,741 bp of genomic sequence and consists of 15 exons. Moreover, we discovered splice variants of the fly OPA1 Technical Assistants mRNA similar to the transcripts generated by the splicing hot Iska Liebner spots in humans and mice.

As part of a large-scale approach to study gene function in Drosophila, P-element insertion lines have been created. In one of these lines, a P-element happened to be inserted into exon 2 of the Drosophila OPA1 gene leading to the functional disruption of this chromosomal gene allele. First studies of this fly line revealed that, while heterozygous flies are see- mingly perfectly viable, homozygous animals die at a late larval stage. Transgenic flies that are being created in our lab will help to dissect the importance of OPA1 function for the nervous system in comparison to other tissues.

Selected Publications

Delettre C, Lenaers G, Pelloquin L, Belenguer P, Hamel C P (2002). OPA1 (Kjer Type) Dominant Optic Atrophy: A Novel Mitochondrial Disease. Mol. Genet. Metabol. 75, 97-107.

Thiselton DL, Alexander C, Taanman JW, Brooks S, Rosen- berg T, Eiberg H, Andreasson S, Van Regemorter N, Munier FL, Moore AT, Bhattacharya SS, Votruba M (2002). A comprehensive survey of mutations in the OPA1 gene in patients with autosomal dominant optic atrophy. Invest Ophthalmol Vis Sci. 43(6):1715-24.

Aung T, Ocaka L, Ebenezer ND, Morris AG, Krawczak M, Thiselton DL, Alexander C, Votruba M, Brice G, Child AH, Francis PJ, Hitchings RA, Lehmann OJ, Bhattacharya SS (2002). A major marker for normal tension glaucoma: association with polymorphisms in the OPA1 gene. Hum Genet. 110(1):52-6.

Pesch, UE, Leo-Kottler, B, Mayer, S, Jurklies, B, Kellner, U, Apfelstedt-Sylla, E, Zrenner, E, Alexander, C, Wissinger, B. (2001). OPA1 mutations in patients with autosomal dominant optic atrophy and evidence for semi-dominant inheritance. Hum. Mol. Genet.10(13):1359-68. 151

Neuronal Stem Cells genesis and activity-dependent cellular plasticity to occur. Our in vivo studies have shown that in the dentate gyrus alone, four distinct cell types with progenitor properties can Gerd Kempermann be identified. We would like to know during which of these stages the actual fate choice decision for neuronal development is made and how it is regulated. We have also characterized other stages of neuronal development in the adult hippocampus. Once the immature neurons have left the cell cycle, they go through a transient stage, during which they express the calcium binding protein calretinin. Most likely, this expression marks the important developmental phase during which the new neurons extend their axon and establish synaptic contacts. Over a period of several weeks, the new cells mature into their ability to respond to a synaptic stimulation and thus become functional as neurons.

Searching for the regulatory master genes

Comparative studies in different strains of mice have revealed that adult hippocampal neurogenesis is regulated on several regulatory levels, which are differentially influenced by inhe- The adult hippocampus, a brain region centrally involved in rited traits. In a linkage study in sixty strains of mice, we learning and memory processes, produces new neurons searched for key gene loci associated with the baseline level throughout life. This “adult neurogenesis” is regulated in an of adult hippocampal neurogenesis in mice. Interestingly, we activity-dependent manner and originates from neuronal stem found that the baseline level of neurogenesis was correlated cells. We are interested in the biology of neuronal stem and with parameters describing the acquisition of a hippocampus- progenitor cells in the adult brain and their contribution to dependent learning task. We identified three candidate loci on brain function in health and disease. We would like to show two chromosomes and are currently trying to identify candi- how new neurons contribute to normal hippocampal function date genes in these regions. and if and how a failure of adult neurogenesis could be involved in the pathogenesis of complex disorders such as memory The research group “Neuronal stem cells” works in close in- loss, temporal lobe epilepsy, major depression, or Alzhei- teraction with the independent group “Neurogenic permissi- mer’s disease. Our hypothesis is that adult neurogenesis and veness”, also headed by Gerd Kempermann, funded by Volks- its activity-dependent regulation allow the brain to optimize wagenStiftung and located at the Dept. of Neurology, Charité the strengths of the mossyfiber tract, an important pathway in the hippocampus, according to functional needs. Beyond these hippocampus-specific aims, we can learn from adult hippocampal neurogenesis about how the development of The putative stem cells of the adult hippocampus have a surprising, tree-like morphology (green). This appearance resembles radial glia, which plays a fundamental role dur- new neurons is possible under the conditions of the adult ing embryonic brain development. The cells that are labeled blue in the microscopic brain. Except for the hippocampus and the olfactory system, image fall into two categories. Some of them have only short processes: these are progenitor cells that are still proliferative. The other type are immature neurons that have the adult brain does not promote the generation of new neu- already extended their dendritic process towards the molecular layer of the dentate rons. This is one reason why many neurological disorders are gyrus - the same area where we find the delicate arborization of the radial glia-like stem chronic and incurable. Our central research questions are cells. what makes the hippocampus a neurogenic region and how can we define this neurogenic permissiveness on a cellular and molecular level.

Characterizing stem cells and neuronal development in the adult brain

Knowledge about the identity, the potential, and the function of neuronal stem cells is still scarce. We have found that the putative stem cells of the hippocampus show glial features, thereby relating them to radial glia and astrocytes. However, the population of proliferating cells that is affected by stimuli regulating adult neurogenesis is a distinct group of progenitor cells lacking glial features and showing among itself a high degree of heterogeneity. Whereas the stem cells carry the general regenerative potential, it might be that the regulation of other progenitor cell populations actually allows neuro- 152

University of Medicine Berlin. The goal of this interaction is to allow clinicians to pursue stem cell research in close interaction with the clinic, but within the scientific environment provided by the MDC.

Selected Publications

Kronenberg, G., Reuter, K., Steiner, B., Brandt, M.D., Jessberger, S., Yamaguchi, M., Kempermann G. (2003). Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli. Journal of Comparative Neurology 467, 455-463.

Brandt, M., Jessberger, S., Steiner, B., Kronenberg, G., Reuter, K., Bick-Sander, A., von der Behrens, W., Kemper- mann, G. (2003). Transient calretinin-expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice. Molecular and Cellular Neuroscience 24, 603-613.

Ehninger, D., Kempermann, G. (2003). Regional differences of wheel running and environmental enrichment on cell genesis and microglia proliferation in the adult murine neocortex. Cerebral Cortex 13, 845-851.

Filippov, V., Kronenberg, G., Pivneva, T., Reuter, K., Steiner, B., Wang, L.P., Yamaguchi, M., Kettenmann, H., Kemper- mann, G. (2003). Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes. Molecular and Cellular Neuroscience 23, 373-382.

Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., Gage, F.H. (2003). Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice. Development 130, 391-399.

Structure of the Group

Group Leader Dr. Gerd Kempermann

Scientists Dr. Gudrun Lutsch Dr. Golo Kronenberg Dr. Anika Bick-Sander Dr. Barbara Steiner Dr. Sebastian Jeßberger Dr. Susanne Wolf Dr. Gerold Brüning (visiting scientist)

Graduate and Undergraduate Students Technical Assistants Dan Ehninger Irene Thun Moritz Brandt Daniela Gast Ana Garcia Ruth Segner Harish Babu Ulrike Ziegler Kathrin Lehmann Erika Kotitschke Benedikt Römer Frank Rolfs 153

Bioethics and Science Online-Conference Communication An Online-Conference of relevant stakeholders was held from September to November 2003 and involved discussion Christof Tannert of the social, ethical, and economic aspects of the research. Its aim was to (1) pilot the use of internet based dialogue forums and (2) to engage stakeholders in a meaningful exchange about the implications of the future of stem cell research. The interface between basic research and its clinical application was highlighted as an important area for further research. The role of the clinician in steering the transfer of research findings into clinical best practice was seen as a key aspect.

Citizens’ Conference

A Citizens’ Conference was held in March 2004 following the Danish model of consensus conferences. A group of 15 lay people was randomly selected to collaborate and produce a “Citizens’ Vote” on the social and ethical aspects of stem cell research. On two weekends in 2003/2004, the group intensively worked on the relevant subject areas to come up with a Project Scope list of questions. These were presented to a group of thirteen experts from science, ethics, policy, and business in a public The interdisciplinary working group, funded by the BMBF expert hearing at the Berlin-Brandenburg Academy of the and the MDC, in co-operation with the Forschungszentrum Sciences. On the basis of the hearing and their previous work, Jülich (FZJ), analyses and develops the societal discourse on the group wrote a statement and presented it to the President biomedicine. Two main aspects form the center piece of the of the Deutsche Bundestag, Wolfgang Thierse. work: (1) The ethical questions that arise in the context of biomedical research and (2) communication of risks and benefits associated with biomedicine. We aim to improve Internet Presence communication between science, politics, media, and the public as well as help to openly debate critical questions The Interactive website (www.bioethik-diskurs.de) was about the opportunities and risks associated with biomedi- established in October 2002 and is continually updated. A cine. We examine how the preconditions for “force (of the barrier-free version is available for disabled persons. Monthly better argument) without coercion” can be created. This visitor numbers average around 2,200 (November 2003). includes the comprehensibility and completeness of scientific-technical information, the clarification of uncertainties within risk, and opportunity assessments as well as the build- Selected Publications ing of trust on the basis of a fair and open dialogue. J. Niewöhner, P. Wiedemann, C. Karger, S. Schicktanz, C. Relevant Topics: Cloning, the creation and use of embryonic Tannert: “Participatory prognosis in Germany – developing stem cells, gene therapy, and the changing notion of disease citizen scenarios for the relationship between biomedicine and illness. and the economy in 2014” (in press) J. Technological Fore- casting and Social Change

Delphi Study A. Görsdorf und C. Tannert: “Die Szenario-Methode als Ver- fahren der Bürgerbeteiligung? Stärken, Schwächen, Möglich- A study on the Future of Stem Cell Research in Germany, re- keiten und Problemquellen aus Sicht von Teilnehmern des ferred to as the ‘Delphi Study’, was conducted. Following Workshops ‘Die Grenzen von Genen, Geld und Gelehrten’” standard procedure, a postal questionnaire with 57 hypotheses (in press) Zschr. Biopolitik on the future of basic and applied research on embryonic and adult stem cells as well as relevant societal aspects was S. Schicktanz, C. Tannert und P. Wiedemann (Eds.): “Kultu- administered twice to leading German stem cell experts. relle Aspekte der Biomedizin: Bioethik, Religionen und All- These Experts had to assess the timeframe of realisation of tagsperspektiven” Campus-Verlag FfM (2003) Series Edition each hypothesis, the respective risks and opportunities for “Kultur der Medizin”, 304 pages, ISBN 3-593-37388-2 patients, research and industry, as well as the most relevant factors influencing the advancement of research in Germany. C. Tannert: “Biologische und theologische Anmerkungen zum The study aims to show trends and time horizons as well as deutschen Ethik-Diskurs zur Biomedizin” (2003) Zschr. Bio- basic parameters and important local/national conditions for politik 2(3), 185 stem cell research. The results will be published in Spring 2004. 154

S. Domasch: “Zukunfts-Szenarien. Szenario-Workshop.” (2003) GenomXpress 1(03),18

S. Domasch und C. Tannert: “Transparenz und Erfahrbarkeit von Wissen – Bioethik und Wissenschaftskommunikation.” (2002) Zschr. Humanontogenetik 5(2), 82

Structure of the Group

Group Leader Dr. Christof Tannert

Collaborating Group Leader (FZJ) Dr. Peter Wiedemann

Scientists Dr. Susanne Reif Dr. Jörg Niewöhner

Associate Scientists Dr. Silke Schicktanz Judith Simon

Guest Scientists Prof. Dr. Philippe Meyer Dr. Norbert Paul

Management Assistant Ali ben Salem

Secretarial Office Ulrike Zehlike

Student Support Lars Kaufmann Mark Schweda Academics 156

Academic Appointments Berufungen

Appointments at the MDC/Joint Appointments Berufungen an das MDC/Gemeinsame Berufungen

The MDC has established an official cooperation agreement Das MDC hat mit der Humboldt-Universität zu Berlin und with the Humboldt University Berlin and the Free University der Freien Universität Berlin Kooperationsverträge abge- Berlin which permits joint appointments. Many of the scien- schlossen, die gemeinsame Berufungen erlauben. Viele der tists appointed to the MDC are interested in a joint appoint- an das MDC berufenen Wissenschaftlerinnen und Wissen- ment with one of the universities of Berlin. Through this schaftler sind an einer gemeinsamen Berufung mit einer der academic link, they wish to participate actively in teaching as Berliner Universitäten interessiert. Sie möchten durch diese well as ensure access to the Berlin universities for their akademische Anbindung aktiv an der Lehre teilnehmen, den Masters and PhD students. The MDC and the Berlin univer- Zugang für ihre Diplomanden/Diplomandinnen bzw. Dokto- sities, likewise, open up the possibility to their employees to randen/Doktorandinnen zu den Berliner Universitäten sichern do doctoral studies and to qualify as lecturers and professors und ihren Mitarbeiterinnen und Mitarbeitern die Möglichkeit in the corresponding Faculties. eröffnen, an den entsprechenden Fakultäten zu promovieren und sich zu habilitieren. Since 2002, when the 5th Amending Act of the Framework Law Governing Universities (Hochschulrahmengesetz) went Seit 2002 kann das MDC auf Grundlage des 5. Änderungsge- into effect, the MDC has been able to appoint junior profes- setzes zum Hochschulrahmengesetz gemeinsam mit Berliner sors jointly with the Berlin universities. In respect to the Universitäten Junior-Professoren berufen. Auf der Grundlage collaboration between the MDC and the Humboldt Univer- der Vereinbarung über die Zusammenarbeit zwischen dem sity Berlin in 1994, a Supplementary Agreement was conclu- MDC und der Humboldt-Universität zu Berlin aus dem Jahre ded in 2002 which allows for the appointment of junior pro- 1994 wurde 2002 eine Ergänzungsvereinbarung abgeschlos- fessors similar to the guidelines for joint appointments to sen, die die Ernennung von Junior-Professoren analog der conventional professorships. Likewise, the MDC signed a Leitsätze für gemeinsame Berufung auf konventionelle Pro- Supplementary Agreement with the Free University Berlin in fessuren ermöglicht. Das MDC hat im Dezember 2002 in December 2002. As a result, the first joint advertisement for gleicher Weise mit der Freien Universität Berlin eine Ergän- junior professorships in the field of Medical Genomics was zungsvereinbarung beschlossen. Eine erste gemeinsame Aus- published in the Spring of 2003. Thus, for the first time, two schreibung auf Junior-Professuren im Bereich Medical Geno- junior professors, Prof. Dr. Michael Gotthardt and Prof. Dr. mics wurde im Frühjahr 2003 veröffentlicht. Mit Prof. Dr. Norbert Hübner, were appointed during the period of the Michael Gotthardt und Prof. Dr. Norbert Hübner konnten report. erstmalig zwei Junior-Professoren berufen werden.

In addition, the following academic appointments were Folgende Wissenschaftler sind berufen worden: made: Prof. Dr. Carmen Birchmeier-Kohler, MDC-Forschungs- Prof. Dr. Carmen Birchmeier-Kohler, MDC Head of the Re- gruppenleiterin in Entwicklungsbiologie/Signaltransduktion search Group Developmental Biology/Signal Transduction in in Nerven und Muskelzellen, hat 2002 den Ruf des MDC und Nerve and Muscle Cells, accepted the offer from the MDC der Freien Universität Berlin auf die C4-S-Professur Mole- and the Free University Berlin of a C4-S-Professorship in kulare Therapie, Signaltransduktion, Entwicklungsbiologie Molecular Therapy, Signal Transduction, and Developmental angenommen. Biology in 2002. 157

Prof. Dr. Thomas Dandekar, MDC-Forschungsgruppe Bio- informatik (Prof. Jens Reich), hat im Dezember 2002 einen Ruf auf den Lehrstuhl für Bioinformatik von der Julius-Ma- ximilians-Universität Würzburg angenommen.

Dr. Yasuyuki Fujita, MDC-Forschungsgruppe Epitheliale Differenzierung, Invasivität und Metastasierung (Prof. Wal- ter Birchmeier), hat im Oktober 2002 einen Ruf als Nach- wuchsgruppenleiter vom Medical Research Council (MRC) Laboratory for Molecular Cell Biology (University College London) angenommen.

Dr. rer. nat. Jörg Hülsken, MDC-Forschungsgruppe Epithe- liale Differenzierung, Invasivität und Metastasierung (Prof. Walter Birchmeier), hat im Januar 2003 einen Ruf als Nach- wuchsgruppenleiter vom Schweizerischen Institut für Experi- mentelle Krebsforschung (ISREC) in Lausanne, Schweiz angenommen.

Prof. Dr. Jens Jordan hat im September 2003 einen Ruf auf die C3-Professur als Leiter des Clinical Research Center an die Charité-Universitätsmedizin Berlin angenommen.

Prof. Dr. Ralph Kettritz, Franz-Volhard-Klinik für Herz- Kreislauferkrankungen, Charité/Helios Klinikum Berlin, Nephrologie und Hypertensiologie, hat im Dezember 2003

„ohne Titel“, 1996, Christine Krämer einen Ruf auf die C3-Professur für Nephrologie der Charité- Copyright: MDC, Photograph: Gunter Lepkowski Universitätsmedizin Berlin angenommen. “Without Title”, 1996, Christine Krämer Copyright: MDC, Photographer: Gunter Lepkowski Prof. Dr. Young-Ae Lee, Zentrum für Genkartierung des MDC und Kinderklinik der Charité der Humboldt-Universität zu Berlin, hat im November 2002 den Ruf auf eine Juniorprofes- sur des Faches Kinderheilkunde an der Charité angenommen. Prof. Dr. Lee’s Forschungsgruppe ist am MDC angesiedelt. Prof. Dr. Thomas Dandekar, MDC Research Group Bioin- formatics (Prof. Jens Reich), accepted a chair of Bioinfor- Prof. Dr. Gary Lewin, Leiter der MDC-Forschungsgruppe matics in December 2002 at the Julius Maximilians Univer- Wachstumsfaktoren und Regeneration, hat im Sommer 2003 sity Würzburg. den Ruf des MDC und der Freien Universität Berlin auf eine C4-S-Professur angenommen. Dr. Yasuyuki Fujita, MDC Research Group Epithelial Differentiation, Invasion, and Metastasis (Prof. Walter Prof. Dr. Stefan Schuster, MDC-Forschungsgruppe Bioinfor- Birchmeier), accepted an offer as a junior group leader at the matik (Prof. Jens Reich), hat im April 2003 einen Ruf auf eine Medical Research Council (MRC) Laboratory for Molecular C3-Professur für Bioinformatik an die Universität Jena ange- Cell Biology (University College London) in October 2002. nommen.

Dr. rer. nat. Jörg Hülsken, MDC Research Group Epithelial Prof. Dr. med. Arya Mitra Sharma, Franz-Volhard-Klinik für Differentiation, Invasion, and Metastasis (Prof. Walter Herz-Kreislauferkrankungen, Charité/Helios Klinikum Ber- Birchmeier), accepted an offer from the Swiss Institute for lin, MDC-Forschungsgruppenleiter Adipositas und Hyper- Experimental Cancer Research (Schweizerisches Institut für tonie, hat Ende 2002 einen Ruf der McMaster University, Experimentelle Krebsforschung (ISREC)) in Lausanne, Swit- Hamilton/Ontario, Kanada angenommen. zerland in January 2003. Prof. Dr. Wolfgang Uckert ist 2002 auf die C3-S-Professur Prof. Dr. Jens Jordan was appointed as a C3 Professor as the Molekulare Zellbiologie und Gentherapie der Mathematisch- Head of the Clinical Research Center at the Charité Univer- Naturwissenschaftlichen Fakultät der Humboldt-Universität sity Medical School and Hospitals Berlin in September 2003. zu Berlin berufen worden. Er ist Nachfolger von Prof. Michael Strauss, der 1999 verstorben ist. Prof. Uckerts wis- Prof. Dr. Ralph Kettritz, Franz Volhard Clinic for Cardiovas- senschaftliche Arbeitsgruppe ist am MDC angesiedelt. cular Diseases, Charité/Helios Teaching Hospitals Berlin, Nephrology and Hypertensiology, accepted a C3-Professor- ship of Nephrology at the Charité University Medical School Berlin in December 2003. 158

Prof. Dr. Young-Ae Lee, MDC Gene Mapping Center and Children`s Hospital of the Charité of the Humboldt-Univer- sity of Berlin, accepted a junior professorship in pediatrics at the Charité in November 2002. Prof. Dr. Lee’s laboratory is located at the MDC.

Prof. Dr. Gary Lewin, Head of the MDC Research Group Growth Factor and Regeneration accepted the offer from the MDC and the Free University of Berlin of a C4-S-Professor- ship of Medical Genome Research in the Summer of 2003.

Prof. Dr. Stefan Schuster, MDC Research Group Bioinforma- tics (Prof. Jens Reich), accepted an offer of a C3-Professor- ship of Bioinformatics at the University of Jena in April 2003.

Prof. Dr. med. Arya Mitra Sharma, Franz Volhard Clinic for Cardiovascular Diseases, Charité/Helios Teaching Hospitals Berlin (Head of MDC Research Group Obesity and Hyper- tension), accepted an offer from the McMaster University, Hamilton/Ontario, Canada at the end of 2002.

Prof. Dr. Wolfgang Uckert was appointed to the C3-S-Profes- sorship Molecular Cell Biology and Gene Therapy in 2002. He is the successor to Prof. Michael Strauss, who died in 1999. Prof. Uckert’s research group is located at the MDC.

Prof. Dr. Rüdiger von Harsdorf, Franz Volhard Clinic for „ohne Titel“, 1996, Christine Krämer Cardiovascular Diseases, Charité/Helios Teaching Hospitals Copyright: MDC, Photograph: Gunter Lepkowski Berlin, and MDC Research Group Molecular Basis of Conge- “Without Title”, 1996, Christine Krämer Copyright: MDC, Photographer: Gunter Lepkowski stive Heart Failure, accepted a C3-Professorship for the spe- cialist field Internal Medicine with a concentration in Cardio- logy at the Charité at the end of 2002.

Prof. Dr. Martin Zenke, MDC Research Group Molecular and Cell Biology of Hematopoietic Cells, was appointed as C4 Professor of Cell Biology and Director of the Helmholtz In- stitute for Biomedical Technology at the University Teaching Hospitals of the Rheinisch-Westfälischen Technischen Hoch- schule (RWTH) Aachen in November 2003. Prof. Dr. Rüdiger von Harsdorf, Franz-Volhard-Klinik für Herz-Kreislauferkrankungen, Charité/Helios Klinikum Ber- lin und MDC-Forschungsgruppe Molekulare Grundlagen der Herzinsuffizienz, hat Ende 2002 den Ruf der Charité auf eine C3-Professur für das Fachgebiet Innere Medizin mit Schwer- punkt Kardiologie angenommen.

Prof. Dr. Martin Zenke, MDC-Forschungsgruppe Molekular- und Zellbiologie hämatopoietischer Zellen, hat im November 2003 den Ruf auf die C4-Professur für Zellbiologie und als Direktor am Helmholtz-Institut für Biomedizinische Techno- logie am Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule (RWTH) Aachen angenommen. 159

Awards/Preise 2002–2003 Jung-Bum Shin 1. Preis des MDC- und FMP-Doktorandensymposiums

Roger Y. Tsien Max-Delbrück-Medaille

Gerd Wallukat Apherese Innovationspreis 2002 der Hans-und-Marlies Stock- Stiftung

2003

Katharina Baum und Gesine Gunkel Abiturientenstipendium des Max-Delbrück-Centrums für Molekulare Medizin (MDC) Berlin-Buch und der Delbrück’ schen Familienstiftung

Boris Engels 1. Preis des MDC- und FMP-Doktorandensymposiums

Detlev Ganten Chevalier de la Legion d`Honneur 2002 Maik Gollasch Franz-Volhard-Preis der Gesellschaft für Nephrologie Andreas Bembenek Young Investigator’s Award for the Sentinel Node Congress Ralph Kettritz Hans-U-Zollinger-Preis der Gesellschaft für Nephrologie Marion Bimmler Bundesverdienstkreuz Friedrich Luft Anthony Raine Award Carmen Birchmeier-Kohler Björn Folkow Preis Gottfried Wilhelm Leibniz-Preis der Deutschen Forschungs- gemeinschaft Ronald McKay Max-Delbrück-Medaille Jana Bröcker und Janine Conrad Abiturientenstipendium des Max-Delbrück-Centrums für Peter M. Schlag Molekulare Medizin (MDC) Berlin-Buch und der Delbrück’ Wilhelm-Warner-Preis für Krebsforschung schen Familienstiftung Clemens A. Schmitt Jan Hinrich Bräsen Kind-Philipp-Preis für Leukämieforschung Andreas-Grüntzig-Forschungspreis

Ralf Dechend Adalbert Bunding-Preis

Detlev Ganten Prof. Ronald McKay vom US-Institut für neurologische Erkrankungen und Schlaganfall (Bethesda), Preisträger der Max-Delbrück-Medaille 2003 Treviranus-Medaille des Verbandes deutscher Biologen Copyright: BioTop/Photo: Agentur Bildschön Prof. Ronald McKay from the National Institute of Neurological Disorders and Stroke Brenda Gerull (NINDS), Bethesda/USA, Recipient of the Max-Delbrück-Medal 2003 Copyright: BioTop/Photo: Agentur Bildschön Oskar-Lapp-Forschungspreis

Michael Gotthardt Sofja Kovalevskaja-Preis der Alexander von Humboldt- Stiftung

Franziska Jundt und Katrin Hoffmann Rudolf-Virchow-Forschungspreis der Charité

Dominik N. Müller New Investigator Award for European Fellows Dieter-Klaus-Förderpreis für Bluthochdruckforschung

Peter M. Schlag Theodor-Brugsch-Preis des Vereins der „Freunde und Förde- rer der Berliner Charité e.V.“ 160

Helmholtz Fellows Helmholtz-Stipendiaten

Helmholtz-Stipendien sind am MDC eingerichtet, um die frühe Unabhängigkeit junger, erfolgversprechender Wissen- schaftler zu ermöglichen. Sie sind für Wissenschaftler vorgesehen, die bereits nachgewiesen haben, dass sie hervorra- gende eigenständige, wissenschaftliche Arbeit leisten.

Die Helmholtz-Stipendiaten sollen an bestehende Arbeits- gruppen des MDC Berlin-Buch angegliedert werden; in diesem Rahmen wird ihnen Raum und Infrastruktur sowie ein Sachmittelbudget zur Verfügung gestellt. Die gastgebende Forschungsgruppe garantiert dem Stipendiaten/der Stipen- diatin thematische Unabhängigkeit; die selbstständige Ein- werbung von Drittmitteln wird erwartet. Das Stipendium wird in der Regel für eine Laufzeit von drei bis fünf Jahren Helmholtz-Stipendiaten (v. l.): Dr. Stefan Britsch, Dr. Katrin Stade, Dr. Jens Jordan, Dr. Armin Rehm, Dr. Brenda Gerull, Dr. Martin Bergmann, Dr. Daniel Krappmann gewährt. Copyright: MDC/Photograph: Uwe Eising Helmholtz-Fellows (from left): Dr. Stefan Britsch, Dr. Katrin Stade, Dr. Jens Jordan, Bewerben können sich Postdoktoranden auf Empfehlung ei- Dr. Armin Rehm, Dr. Brenda Gerull, Dr. Martin Bergmann, Dr. Daniel Krappmann Copyright: MDC/Photographer: Uwe Eising nes Forschungsgruppenleiters aus dem MDC Berlin-Buch beim MDC-Vorstand. Es folgt dann eine Begutachtung in den MDC-Gremien. Im Berichtszeitraum hat das MDC insgesamt 12 Stipendiaten gefördert.

Helmholtz Fellowships at the MDC are intended to allow promising young scientists to carry out their own independent research. Helmholtz Fellows have demonstrated that they are capable of conducting high quality research.

Fellows are associated with MDC host research groups and, therefore, receive lab space, infrastructure, and a research budget. The host research group guarantees the Fellow’s in- dependence in terms of research topic. In addition to MDC support, Fellows are expected to apply for external funding sources. Fellowships are typically granted for between three and 5 years.

Eligible are post-doctoral scientists with a strong recommendation from an MDC group leader. Applications are received by the MDC Scientific Director and reviewed by a selection committee. During the report period, the MDC supported 12 Helmholtz Fellows. 161

International PhD Program Internationales PhD-Programm

The international PhD program “Molecular Cell Biology” is a Das Internationale PhD-Programm „Molekulare Zellbiolo- joint activity of the Max-Delbrück Center (MDC) for Mole- gie“ ist ein Gemeinschaftsprojekt des Max-Delbrück-Cen- cular Medicine and the Humboldt University (HU) Berlin. trums für Molekulare Medizin (MDC) Berlin-Buch und der The program provides training and research opportunities for Naturwissenschaftlichen Fakultät der Humboldt-Universität university graduates who wish to obtain a PhD in the fields of (HU) zu Berlin. Es wendet sich an Hochschulabsolventen, die Cell Biology, Molecular Biology, Molecular Genetics, Mole- eine Dissertation auf dem Gebiet der Zellbiologie, Moleku- cular Cardiovascular Research, Cancer Research, Develop- larbiologie, Molekulargenetik, der molekularen Herz-Kreis- mental Biology, and Neurobiology. Training and research lauf sowie Krebsforschung, Entwicklungsbiologie oder Neu- within the PhD program is interdisciplinary with strong links robiologie anfertigen wollen. Ausbildung und Forschung in between basic research and medicine. diesem PhD-Programm sind interdisziplinär, wobei eine intensive Verbindung zwischen Grundlagenforschung und Me- Students write a research proposal in the first year and give dizin im Vordergrund steht. annual presentations of their progress in the following years. Students are advised by their Research Group Leader and two Die Studenten verfassen im ersten Jahr einen Forschungsan- advisors of their PhD Committee and obtain their PhD degree trag und in jedem folgenden Jahr eine Darstellung ihrer Fort- after approval through the Humboldt University (Dr. rer. nat.) schritte. Sie werden durch ihren Forschungsgruppenleiter or through their national university. und zwei Berater des PhD-Ausschusses unterstützt. Ihren akademischen Grad erhalten sie von der Humboldt-Univer- Eligible are students who have obtained an academic degree sität (Dr. rer. nat.) oder der jeweiligen Universität ihres Hei- comparable to the Masters degree or to the German Diploma. matlandes. Admission to the MDC-HU PhD program is competitive and decided by the Graduate Committee. Financial support via Teilnahmeberechtigt sind Studenten, die bereits einen akade- PhD fellowships is provided by the MDC. mischen Grad, vergleichbar mit dem „Master“ oder dem deutschen „Diplom“ besitzen. Die Zulassung zum Internationalen During 2003, the MDC received approximately 200 applica- PhD-Programm wird in einem kompetitiven Auswahlverfah- tions and accepted 9 applicants into the program. ren durch den Graduiertenausschuss des MDC ermittelt.

2003 erhielt das MDC rund 200 Bewerbungen, 9 Bewerber sind in das Programm aufgenommen worden. Sie erhalten alle ein Stipendium des MDC. 162

Congresses and Scientific Meetings

Kongresse und Wissenschaftliche Tagungen

2002 Tumorkonferenz, Helios Klinikum (September 28, 2002) “8th Liposome Research Days Conference” Max-Delbrück-Centrum für Molekulare Medizin (MDC) “ISGO Conference on Structural Genomics 2002” Berlin-Buch International Structural Genomics Organisation (ISGO) und (May 21–24, 2002) Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch Onkologische Fachtagung (October 10–13, 2002) Deutscher Berufsverband für Pflegeberufe mit Robert-Rössle-Klinik “Berlin Lecture on Molecular Medicine” (June 5–7, 2002) Prof. Roger Tsien, Howard Hughes Medical Institute, University of California San Diego (USCF) “Lange Nacht der Wissenschaften” auf dem Campus Berlin- “Imaging Signal Transduction and Protein Sociology” Buch (November 21, 2002) (June 15, 2002) “Molecular Biosensors in Neuroscience” “Wissenschaft macht Schule” Max-Delbrück-Centrum für Molekulare Medizin (MDC) Eine Vortragsreihe für Schüler mit Besuch der interaktiven Berlin-Buch Mikroskopausstellung (November 21–22, 2002) Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch, (June 24–28, 2002) 2003

PhD-Symposium “Genomics and proteomics based therapy strategies” Max-Delbrück-Centrum für Molekulare Medizin (MDC) Partnering Workshop between France and Bioregion Berlin-Buch Berlin-Brandenburg (July 1, 2002) (February 17, 2003)

Laien-Experten-Scenario der AG Bioethik und “Anwendung von Imaging und Patch Clamp in den Wissenschaftskommunikation des Max-Delbrück-Centrums Zellulären Neurowissenschaften” für Molekulare Medizin (MDC) Berlin-Buch Max-Delbrück-Centrum für Molekulare Medizin (MDC) (September 26–28; October 19; November 7–9, 2002) Berlin-Buch (May 17, 2003) “Ernst Schering Lecture 2002” Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch und Schering Forschungsgesellschaft Ian Wilmut: “Cloning in Biology and Medicine” (September 26, 2002) 163

“Berlin-Buch Congress of Biotechnology” “Euroglia 2003 – VI. European Meeting on Glial Cell BioTOP Berlin-Brandenburg Function in Health and Disease” Max Delbrück-Centrum für Molekulare Medizin (MDC) Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch Berlin-Buch Friedrich-Ebert-Stiftung (FES) (September 3–6, 2003) Berlin-Buch Management GmbH (BBM) Bundesverband der Pharmazeutischen Industrie (BPI) “Common Mechanisms in Development and Cancer” Berliner Wirtschaftsgespräche Max-Delbrück-Centrum für Molekulare Medizin (MDC) (June 13–14, 2003) Berlin-Buch (September 26, 2003) “Lange Nacht der Wissenschaften” auf dem Campus Berlin-Buch 7. Chirurgische Forschungstage (June 14, 2003) Robert-Rössle-Krebs-Klinik, Charité/Helios Klinikum Berlin (October 16–18, 2003) 13. Symposium zu Leukämie- und Lymphomdiagnostik mit Mikroskopie- und Durchflußzytometrie-Kurs “3rd International Symposium on Obesity and (“Kieler Symposium”), Hypertension” Robert-Rössle-Krebs-Klinik Charité, Campus Berlin-Buch/ Franz-Volhard-Clinic for Cardiovascular Diseases, Charité/ Helios Klinikum Berlin Helios Klinikum Berlin (June 18–21, 2003) (October 23–25, 2003)

PhD Symposium 2. Bucher Hämatologie-Forum Max-Delbrück-Centrum für Molekulare Medizin (MDC) Beckman Coulter GmbH Berlin-Buch Robert-Rössle-Krebs-Klinik, Charité/Helios Klinikum Berlin (June 26, 2003) (November 7–8, 2003)

“Berlin Lecture on Molecular Medicine” Prof. Ronald McKay, National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, USA “Stem Cells in Science and Medicine” (December 11, 2003) Mit dem frisch gekürten Chemienobelpreisträger Prof. Kurt Wüthrich (re.) von der Eid- genössischen Technischen Hochschule (ETH) Zürich, Schweiz, und Chemienobelpreis- träger Prof. Robert Huber vom Max-Planck-Institut für Biochemie, Martinsried, waren “Bürgerkonferenz zur Stammzellforschung” gleich zwei Laureaten zur International Conference on Structural Genomics (10.–13. Ok- AG Bioethik und Wissenschaftskommunikation des tober 2002) nach Berlin-Buch gekommen. Copyright: MDC/Photograph: Andreas Knespel Max-Delbrück-Centrums für Molekulare Medizin (MDC) Nobel Prize Winner Prof. Kurt Wüthrich (right) of the Swiss Federal Institute of Techno- Berlin-Buch logy (ETH) Zurich and Nobel Prize Winner Prof. Robert Huber from the Max Planck Institute of Biochemistry, Martinsried attended the International Conference on Structural (December 12–14, 2003) Genomics (Oktober 10–13, 2002) in Berlin-Buch. Copyright: MDC/Photographer: Andreas Knespel 164

Seminars Seminare

2002

Speaker Institute Titel of Seminar

Ariel Achtman National Institute for Medical Research, B cell responses in murine malaria Division of Parasitology, London/UK Scott V.Adams Vanderbilt University Nashville/USA Electrophysiological Investigation of Coupling in the Human Serotonin Transporter Birgit Alpen Cornell University, New York/USA SEREX-analysis in gastric marginalzone B-cell lymphoma of MALT-type Frank Baas Academic Medical Center, Neurogenetics Allele specific inhibition, a novel Laboratory Amsterdam/NL approach to treatment of genetic disease Konrad Basler University Zürich/Switzerland Dpp signalling, cell competition and apoptosis in Drosophila limbs Uta Bauer IMT University of Marburg/Gemany Regulation of gene expression by arginine methylation Etienne Baulieu Collège de France, Le Kremlin-Bicêtre, Neurosteroids: A New Function of the Paris/France Brain. Aging, Memory, Myelination Yinon Ben-Neriah The Lautenberg Center for Immunology, Obstacles en route to degradation: lessons Hebrew-University-Hadassah, Jerusalem/Israel from the NF-kB and Wnt pathways Stefan Berry Ruhr-Universität Bochum/Gemany Biological Electron Transport Systems - Theme and Variations Paola Bezzi Université de Lausanne/Switzerland Prostaglandins mediate a major component component of astrocyte calcium elevations responsible for receptor-stimulated glutamate release Hans Bock UT Southwestern Medical Center Dallas/USA Reelin signaling through lipoprotein receptors - Role of Src family kinases Ulrich Bogdahn Neurologische Universitätsklinik TGF-Beta and malignant gliomas Regensburg/Gemany -from pathogenesis to therapy Jürgen Bolz University of Jena, Institute of Zoology/ Wiring Molecules for the Assembly of Gemany Cortical Circuits 165

Speaker Institute Titel of Seminar

Ralf Brandt Novartis Pharma AG, Basle/Switzerland The Reconstituted Genetically Manipula- ted Mouse Mammary Gland: A novel preclinical animal model for cancer research

Armin Braun Fraunhofer-Institut für Toxikologie, Are neurotrophins a suitable target in Aerosolforschung und Pharmaforschung asthma therapy? Hannover/Germany Tom Brismar Karolinska Hospital, Stockholm/Sweden Physiology of gliomas Jean-François Brunet CNRS UMR 8542, Département de Biologie, Phox2b - master gene of a neural circuit Ecole Normale Supérieure, Paris/France Thorsten Burmester Institute for Zoology, University of Neuroglobin: A respiratory protein of Mainz/Germany the brain Selina Chen-Kiang Cornell University, New York/USA CDK inhibitors: essential controls of B cell terminal differentiation and tumorigenesis Stephen Cohen EMBL Heidelberg/Germany Boundary formation in Drosophila wing development Rudolf A.Deisz Charité University Medicine Berlin/Germany Impaired function of metabotropic GABA receptors in slices from epilepsy surgery Gunnar Dittmar Harvard Medical School/ USA Hub1 - the ubiquitin-like link to cell polarity Veit Flockerzi University Saarland-Saarbrücken, The trp channels, a remarkably Pharmakology and Toxikology/Germany functional family Mikio Furuse Dept. of Cell Biology, Kyoto University/Japan The role of claudins in epithelial barrier function Ali Gorji University of Muenster/Germany Spreading depression in human neocorti- cal slices and rat spinal cord tissues Magdalena Götz Max Planck Institute for Neurobiology Glial cells generate neurons: molecular Munich/Germany and cellular mechanisms of neurogenesis Ingrid Grummt Molecular Biology, DKFZ Regulation of gene expression in Heidelberg/Germany eukaryotes: Lessons learned from RNA polymerase I Francois Guillemot Institut de Génétique et de Biologie, Proneural genes and cell type Moléculaire et Cellulaire C.U. de specification in the murine Strasbourg/France telencephalon Ulrich Hämmerling Memorial Sloan-Kettering Cancer Center, Vitamin A: new life for an ancient New York/USA molecule Christian Harteneck Free University Berlin, Institute for Structure and function of Pharmacology/Germany TRP-homologous channels Jonathan Horsford Department of Molecular and Medical Genetics, Chx10 and Mitf have dynamic and University of Toronto, Ontario/Canada opposing functions in mammalian retinal cell fate decisions Jonathan Howard Max Planck Institute of Molecular Cell Chemical-to-Mechanical Energy Biology and Genetics, Dresden/Germany Transduction by the Motor Protein Kinesin Miho Inoue- Gifu University/Japan Allelic variation of the dog dopamine Murayama receptor D4 gene polymorphic region and its relation to behavioral traits Berend H.Isermann Blood Research Institute, Blood Center Genetically designed mice provide new of Southeastern Wisconsin, Milwaukee/USA insights into the function of the thrombo- modulin - protein C pathway 166

Speaker Institute Titel of Seminar

Herbert Jäckle MPI Göttingen/Germany From egg to embryo: Functional genetics with the model organism Drosphila Ricarda Jahnel Free University Berlin/Germany Biochemical characterisation of the vanil- loid receptor 1 (VR1) Leszek Kaczmarek Nencki Institute, Warsaw/Poland c-Fos/AP-1 to matrix metallo-proteinases, a missing link in neuronal plasticity? Istvan Kiss Biological Research Centre, Hungarian Apart from nuclear import - the role of Academy of Sciences Szeged/Hungary importin-alpha in Drosophila oogenesis Rüdiger Klein Max-Planck-Institute of Neurobiology Ephrins and Eph receptors - bidirectional Martinsried/Germany regulators of development and adult plasticity Jari Koistinaho University of Kuopio/Finland p38 MAPK-mediates increased ischemic vulnerability in beta-APP transgenic mice Tony Kouzarides Wellcome/CRC Institute, Cambridge Histone methylation in transcriptional University/UK control Georg W.Kreutzberg Max-Planck-Institute for Neurobiology, Trial & Error in Microglial Research Martinsried/Germany Kerstin Krieglstein University of Goettingen/Germany TGF - regulators of cell growth and differentiation Stephan Kröger University of Mainz/Germany The synapse-organizing proteoglycan agrin in the developing CNS: expect the unexpected Ulrich Kubitscheck Institut for Experimental Physics, University Insight into the dynamical structure of the of Bremen/Germany cell nucleus by visualization of single molecules and single RNP particles Ralf Kuehn Artemis Pharmaceuticals GmbH New technology to generate KO mice Cologne/Germany Damien Kuffler University of Puerto Rico/South America Promoting regeneratio in the adult human PNS and CNS Luc Leybaert Gent University/Belgium Astrocyte-endothelial calcium talk: a signal at the barrier? Andrew Matus Friedrich Miescher Institut, Basel/Switzerland Actin dynamics at synaptic connections in the brain: mechanisms and possible meanings Petra May UT Southwestern Medical Center Dallas/USA Proteolytic processing of LRP mediates regulated release of its intracellular domain Kevin J.McElwee Department of Dermatology, Philipp Alopecia areata in humans and rodents University Marburg/Germany models Serge Muyldermans Vrieje Universiteit Brussel/Belgium Camel antibodies as reporter molecules Teresa Nicolson Max Planck Institute for Developmental Swimming in circles: genetic dissection Biology Tübingen/Germany of hearing and balance in zebrafish Rienk Offringa Leiden University Medical Center, Role of serine protease inhibitors Netherlands (serpins) in dendritic cell survival and tumor immune escape Thomas Park University of Illinois at Chicago/USA Unique sensory adaptations of the naked mole-rat Michal Pravenec Czech Academy of Science Praha, Institute Genetic analysis of a complex metabolic of Physiology/Czech Republic syndrome in the spontaneously hypertensive rat 167

Speaker Institute Titel of Seminar

Andreas Püschel Westfälische Wilhelms-Universität, Signal transduction by receptors for the Münster/Germany repulsive axon guidance signal sema- phorin 3A: the role of Rho-like GTPases Nikolaus Rajewsky The Rockefeller University, New York/USA Computational approaches for decoding transcriptional regulation Hans-Georg Rammensee University of Tübingen/Germany Towards patient-individual tumor peptide selection for vaccination Ivan Raska Academy of Science Praha, Institute of Searching for active ribosomal genes Experimental Medicine/Czech Republic Dan Roden Vanderbilt University, School of Medicine, The Long QT Syndrome Nashville/USA Klaus Rohr University of Cologne/Germany Development of the thyroid gland in zebrafish Sascha Sauer Max-Planck-Institute for Molecular Genetics Genotyping Single Nucleotide Berlin/Germany Polymorphisms by MALDI Mass Spectrometry Mathias Seeliger University Eye Hospital Tuebingen/Germany Analysis of retinal physiology based on animal models of inherited retinal degenerations Giulio C.Spagnoli University of Basel/Switzerland Induction of CTL against tumor associated antigens in melanoma patients upon immunization with recombinant vaccinia virus Hans Stassen University Hospital Zurich/Switzerland Genetic diversity - quantification and analysis David Stuart Oxford University, Structural Biology Towards the Atomic Structure of an Centre/UK Enveloped Virus: Bacteriophage PRD1 Gerd Sutter GSF München, Institut für Molekulare Immunogenicity and protective capacity Virologie/Germany of vaccinia virus MVA vectors in animal models for viral disease and cancer Bernard Swynghedauw INSERM Paris/France Cardiac remodeling. An evolutionary perspective Dawn Teare University of Sheffield, Mathematical Sampling properties of summary Modelling and Genetic Epidemiology measures of Linkage Disequilibrium Group/UK Roger Tsien Howard Hughes Medical Institute, University Imaging Signal Transduction and Protein of California San Diego/USA Sociology André Uitterlinden Erasmus University Rotterdam, Department Genetics of osteoporosis of Internal Medicine/Netherlands Gunter Weiss MPI für Molekulare Anthropologie, Analyses of primate Xq 13.3 data- Leipzig/Germany from populations to substitution models Ian Wilmut Roslin Institute, Department of Gene Expression Cloning in Biology and Medicine and Development, Edinburgh/Scotland Jürgen Winkler Neurologische Universitätsklinik Functional recovery after hippo- Regensburg/Germany campampal and cholinergic deafferentation Lode Wyns Freie Universität Brüssel/Belgium Arsenate reductase, a phosphotyrosine- phosphatase drafted for redox duty Alpha Yap University of Queensland, Brisbane/Australia Cell-cell recognition: E-cadherin signaling to the actin cytoskeleton Peter Zahradka St. Boniface General Hospital Research Novel targets for antirestenosis Centre, Winnipeg/Canada intervention 168

2003

Speaker Institute Titel of Seminar

Sylvia Arber Universität Basel Molecular pathways controlling the assembly of the spinal monosynaptic reflex circuit Bernd Arnold DKFZ Heidelberg/Germany Tolerance induction by non-professional antigen presenting cells Uri Ashery Dept. of Neurobiochemistry Tel-Aviv Molecular mechanisms of synaptic University/Israel transmission Mathias Bähr University of Göttingen, Neurology/Germany Neuroprotection - models, mechanisms, perspectives Anne Baron-van CHU Pitié-Salpêtrière, Paris/France Neural progenitors and CNS myelin repair Evercooren Konrad Basler Dept. of Structural Biology, Stanford Growth and patterning in Drosophila University School of Medicine/USA limb development Peter B.Becker Ludwig-Maximilians University Targeting of the dosage compensation Munich/Germany complex to the male X chromosome in Drosophila Jürgen Behrens Nikolaus Fiebiger Center for Molecular Control of Wnt Signaling in Medicine Erlangen/Germany Tumorigenesis Philippe Bertolino Intern. Agency for Research on Cancer, Developmental and tumor studies using Lyon, France Men1 knock-out mouse models Axel Borst MPI Martinsried/Germany Neural circuits in the cockpit of the fly Thomas Braun University of Halle, Inst. of Physiological Plasticity of myogenic differentiation: Chemistry/Germany Muscle stem cells in regeneration and development of cardiac and skeletal muscle Jorge Caamano MRC Centre for Immune Regulation Regulation of secondary lymphoid tissue University of Birmingham organogenesis by the NF-kB proteins. Peter Carmeliet University of Leuven and Center for Transgene Genetics in the mouse, humans and Technology & Gene Therapy/Belgium zebrafish to dissect vascular development and disease

Patrick Carroll University of Montpellier/France New perspectives on reelin signalling in neuronal migration Elena Cattaneo University of Milano/Italy The huntingtin syndromes Hans Clevers Dept. Immunology, University Hospital Sculpting the gut Utrecht/Netherlands Pam Cowin Dept. of Cell Biology, New York University Catenins in the control of mammary Medical Center/USA and epidermal proliferation and cell fate Monica di Luca University of Milano/Italy Structural organization of the excitatory synapse: role of MAGUK in the Post- synaptic Density Martin Eilers Philipps University Marburg/Germany Mechanisms of Transcriptional Regulation by Myc Peter Ekblom Cell and Molecular Biology, Lund University, Role of laminin-1 for epithelial Lund/Sweden development Riccardo Fodde Dept. of Human and Clinical Genetics, Leiden Stem cell differentiation, signal trans- University Medical Center/Netherlands duction and genetic instability in colorectal cancer 169

Speaker Institute Titel of Seminar

Stephan Frank Dept. of Neuropathology, University Mitochondria during Apoptosis: Bonn/Germany Divide and Perish ... Clemens Franz Ludwig Institute for Cancer Research, Association of p120 catenin with Martin London, UK microtubules and the centrosome Jonas Frisen Karolinska Institute Stockholm/Sweden Generation of neurons from stem cells in the adult brain Michael Frotscher University of Freiburg/Germany Reelin controls neuronal migration by acting on the radial glial scaffold Craig Garner Stanford University/USA Cellular mechanisms of CNS synaptoge- nesis Max Gassmann University of Zürich/Switzerland Constitutive, HIF-1-independent erythro- poietin gene expression: how transgenic mice cope with a hematocrit of 80% Christo Goridis Ecole Normale Superieure Paris From the control of neurogenesis to autonomic disorders: the role of the Phox2 transcription factors Rudolf Grosschedl Institute for Biochemistry and Genetics/ Wnt-dependent and Wnt-independent University of Munich/Germany regulation of organogenesis by LEF1 Christian Haass University of Munich Intramembraneous Proteolyisis and Alzheimer’s Disease Alison K.Hall Cleveland University/USA Sensory neurons in development and injury: the role of activin Matthias Hammerschmidt Max Planck Institute for Immunobiology, BMPs and partners during embryogenesis Freiburg/Germany and organogenesis of the zebrafish Bernd Heimrich Charité University Medicine Berlin, Regulators of hippocampal development Institute of Anatomy/Germany Carl-Philipp Heisenberg Max Planck Institute for Molecular Cell Molecular and cellular control of cell Biology and Genetics Dresden/Germany migration during zebrafish gastrulation Joachim Herz University of Texas, Southwestern Medical Mechanisms of signaling by ApoE Center/USA receptors in the brain and vascular wall Wolfgang Holnthoner University of Vienna, Division of General Fibroblast growth factor-2 induces Dermatology, Vienna/Austria Lef/Tcf-dependent transcription in human endothelial cells Steve Hunt University College London Substance P in Depression, Addiction and Pain Beat Imhof Department of Pathology, Centre Medical Analysis of adhesion molecules in cell Universitaire Geneva/Switzerland migration Rodney Kellems University of Texas, Dept. of Autoantibodies against the Angiotensin Biochemistry/USA Receptor in Preeclampsia Rolf Kemler Department of Molecular Embryology/ -Catenin controls cell fate in mouse Max-Planck Institute of Immunobiology development Freiburg/Germany Susan Kentner HGF office Brussels/Belgium 6th European Framework Programme, Contract and Budget Issues, Project Management, Proposals and Results from the 1st Call Ralph Kettritz HELIOS Klinikum Berlin/Charité, Mechanisms of ANCA-associated Franz Volhard Clinic, Nephrology and Vasculitis Hypertension/Germany 170

Speaker Institute Titel of Seminar

Christian Klämbt University of Muenster/Germany Genetic Analysis of Neuron-Glia Inter- action at the CNS Midline of Drosophila Wilfried Kraus HGF office Brussels/Belgium 6th European Framework Programme. Contract and Budget Issues, Project Management, Proposals and Results from the 1st Call Michaela Kuhn University of Muenster, Institute of Cell-restricted deletion of the guanylyl Pharmacology and Toxicology/Germany cyclase-A receptor in mice unravels the various physiological functions of atrial natriuretic peptide (ANP) Erich Lanka Max-Planck-Institut für Molekulare Genetik, The role of pro-telomerase in the Berlin replication of linear DNA Urban Lendahl Karolinska Institute, Stockholm/Sweden Notch signaling in development and disease Clotilde Levecque Institute Pasteur de Lille, France Genetic determinants of Parkinson’s disease James K.Liao Harvard Medical School, Vascular Medicine Novel Nongenomic Actions of Steroid Research/USA Hormones on the Vascular Wall Joachim Luebke Research Center JuelichGermany Morphological and functional aspects of excitatory synaptic transmission in the barrel cortex Bernhard Lüscher RWTH Aachen, Institut for Biochemistry The Myc/Max/Mad network: and Molecular Biology/Germany transcriptional control of cell behavior Lamberto Maffei Istituto di Neuroscienze Pisa/Italy Molecular basis of plasticity in the mammalian visual cortex Stefanie Mandl Stanford University, School of Medicine/USA Understanding immune cell trafficking via in vivo bioluminescence imaging Roland Martin NIH Bethesda/USA Novel Findings on the Pathogenesis of Multiple Sclerosis Evelyne May Centre d Energie Atomique Paris/France Recent data on in vitro and in vivo p53- dependent transcriptional activity Frank Mc Cormick University of California, San Francisco/USA Cancer Therapy based on Ras and p53 John McGiff New York Medical College Valhalla, Renal COX-2, Cytokines, and 20-HETE: New York/USA Tubular and Vascular Mechanisms Ronald McKay National Institute of Neurological Disorders Stem Cells in Science and Medicine and Strike (NINDS), Bethesda/USA Barbara Munz Stanford University Medical Center Differential gene expression in myogenic Stanford/USA differentiation Erwin Neher University of Goettingen Dissecting Short Term Synaptic Plasticity at the Calyx of Held Scott Ness Molecular Genetics & Microbiology Complexities of gene regulation by Myb University of New Mexico/USA transcription factors Genevieve Nguyen INSERM U489, Paris/France The renin receptor - A new partner in the renin-angiotensin system Christof Niehrs DKFZ Heidelberg/Germany Molecular mechanism of embryonic head induction Christiane Nüsslein- Max Planck Institute for Developmental Cell migration in zebrafish Volhard Biology Tübingen/Germany Stefan Offermanns University of Heidelberg/Germany GPCRs and plexins in neural development and function 171

Speaker Institute Titel of Seminar

Yoshiaki Ohkuma Osaka University, Laboratory of Cellular Physical and functional links between Biolog/Japan human mediator complexes and general transcription machinery Christopher Payne University Zürich/Switzerland Motor Proteins, Endomembranes and Nuclear Dynamics During Mammalian Fertilization Vladimir Pekarik University of Zürich/Switzerland RNA interference in ovo to study axon guidance Michel Pletschette European Commission Research International Ethical Issues in clinical research and Cooperation, Brussels/Belgium trials in developing countries Lincoln R.Potter University of Minnesota, USA Regulation of and Signaling Through Cyclase-Linked Natriuretic Peptide Receptors Klaus T.Preissner Justus Liebig University Giessen, Institute Trick and Treat: New Endogenous and for Biochemistry/Germany Exogenous Anti-inflammatory Molecules

Nikolaus Rajewsky NYU, New York/USA Computational Decoding of Gene Regula- tory Networks in Multicellular Organisms Christoph Redecker Friedrich-Schiller-Universität Jena, Functional consequences of focal lesions Neurology/Germany in the developing and adult brain Louis Reichardt Howard Hughes Medical Institute, Dept. of Catenin functions in synapse formation Physiology, University of California and function San Francisco/USA Diethelm Richter University of Göttingen/Germany Serotonergic Modulation of the Respiratory Center Frank Rosenbauer Harvard Medical School, Beth Israel Stem Cells, Progenitors, and Deaconess Medical Center, Boston/USA Malignancies: Acute Myeloid Leukemia Induced by Reduction of Transcription Factor PU.1 in Mice Patricia Ruiz Max-Planck Institute for Molecular Genetics, Molecular Approaches to Heart Failure Berlin/Gemany Kathryn Sandberg Georgetown University, Washington/USA Translational control of the angiotensin receptor by alternative splicing Constance Scharff MPI of Molecular Genetics Berlin/Germany Insights from bird brains: pathways for learned vocal communication, regulation of adult neurogenesis, and characterization of a “speech” gene Martina Schinke Harvard Medical School, Boston/USA Cardiogenomics: Identifying gene expression profiles in cardiac hypertrophy Christian A.Schmidt Ernst-Moritz-Arndt Universität Greifswald, Characterization of atypical T-cell- Zentrum für Innere Medizin receptor (TCR) rearrangement Hans Schöler Germline Development Group University From ES cells to Oocytes: A stepwise of Pennsylvania Philadelphia/USA Approach to Nuclear Transplantation Therapy Markus Schülke Charité University Medicine Berlin, Institute Proteome analysis in patients with of Pediatrics/Germany mitochondriopathies Thomas Schwartz Rockefeller University, New York/USA Structure of the eukaryotic SRP-receptor: New insight into the complex regulation of protein targeting Peter Seeburg University of Heidelberg Genetic manipulation of synaptic function in the mouse: Probing synaptic plasticity and spatial learning 172

Speaker Institute Titel of Seminar

Atsuko Sehara- Kyoto University/Japan Roles of Metalloprotease-disintergrin Fujisawa (ADAM)s in morphogenesis

Marjeta Sentjurc Jozef Stefan Institute, Ljubljana/Slovenia Electron paramagnetic resonance (EPR) in the investigation of biological processes Paul Sharpe Kings College London, Dental Institute, Molecular control of tooth patterning: Dept. of Craniofacial Development/UK towards regenerative dentistry Curt Sigmund University of Iowa, Iowa City/USA Dissecting complex physiological pathways using targeted gene expression Mikael Sigvardsson Department for Stem Cell Biology, Co-ordination of gene expression by Lund/Sweden helix-loop-helix transcription factors in early B lymphocyte development Jan A.Staessen Laboratory of Hypertension Campus Essential Hypertension Gasthuisberg Leuven/Belgium Archontoula Stoffel The Rockefeller University, New York/USA API2/MALT1, NF-kB and p53: a combi- national network that promotes lymphomagenesis Max Topp University of Tübingen/Germany Immunotherapy strategies for targeting B-cell malignancies Elmar Wahle University of Halle/Germany Polyadenylation of pre-mRNA: Mecha- nisms of processive elongation and length control Andrew H.J. Wang Institute of Biological Chemistry, Structural proteomics in Taiwan Academia Sinica, Taipei/Taiwan Eva Wardelmann Institute of Pathology, University Bedeutung von c-kit-Mutationen für das Bonn/Germany biologische Verhalten Gastrointestinaler Stromatumoren Bill Weis Albers School, University of Seattle/USA Biochemical and structural analysis of beta-catenin in cell adhesion and Wnt signaling Heiner Westphal NIH, Bethesda/USA Transcriptional control of early mouse development Ralph Willemsen Erasmus MC University Medical Center Genetic engineering of T cell specificity Rotterdam/NL Martina Witsch- Institute of Medical Biology and Human Population Genetics of the Baumgartner Genetics, Innsbruck/Austria Smith-Lemli-Opitz Syndrome Xia Yiang University of Texas, Dept. of Autoantibodies against the Angiotensin Biochemistry/USA Receptor in Preeclampsia Structure and Organization 174

The MDC Berlin-Buch Das MDC Berlin-Buch

The MDC Berlin-Buch and Clinical Research Das MDC Berlin-Buch und die klinische Forschung

Clinical Research Klinische Forschung The Max-Delbrück-Center for Molecular Medicine (MDC) Das Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch makes around five million Euros available from Berlin-Buch stellt jährlich fünf Millionen Euro aus seinem its budget for joint research projects conducted with two spe- Budget für Forschungsvorhaben mit den beiden Spezialklini- cialist clinics of the Charité University Medical School and ken der Charité – Universitätsmedizin Berlin/Helios Klini- Hospital Berlin/The Helios Teaching Hospitals Berlin – kum Berlin – der Franz-Volhard-Herz-Kreislaufklinik (FVK) namely, the Franz-Volhard Cardiovascular Clinic (FVK) and und der Robert-Rössle-Krebsklinik (RRK) zur Verfügung. In the Robert-Rössle Cancer Clinic (RRK). As part of these joint gemeinsamen Forschungsprojekten suchen klinische For- projects, researchers and scientists of the MDC search for scher und Wissenschaftler des MDC nach Genen und Mecha- genes and mechanisms that play a role in diseases such as nismen, die bei Krankheiten wie Bluthochdruck, Fettstoff- high blood pressure, metabolic disorders, cardiovascular wechselstörungen, erblich bedingten Herzvergrößerungen diseases, and cancer. The four clinical directors of the FVK und Krebs eine Rolle spielen. Die vier leitenden Ärzte der and the RRK have their own research group at the MDC. As FVK und der RRK haben gleichzeitig eigene Forschungs- MDC Research Group Leaders, they are automatically mem- gruppen am MDC. Als MDC-Forschungsgruppenleiter sind bers of all MDC committees including the Management sie automatisch Mitglieder in allen MDC-Gremien einschließ- Committee. lich des Leitungskollegiums.

MDC scientists have jointly reached agreement on three Wissenschaftlerinnen und Wissenschaftler des MDC haben structural foci of the collaboration with their clinical partners: sich gemeinsam mit ihren klinischen Partnern auf drei strukturelle Schwerpunkte der Zusammenarbeit verständigt: • Clinical Cooperation Projects (KKP): Here, joint research projects are conducted on the topics of cardiovascular and • Klinische Kooperationsprojekte (KKP): Hier werden metabolic diseases, cancer research as well as the function von Grundlagenwissenschaftlern und Klinikern gemein- and dysfunction of the nervous system. sam Forschungsprojekte zu den Themen Herz-Kreislauf- • Clinical Research Units (CRU): The Clinical Research und Stoffwechsel-Erkrankungen, Krebsforschung sowie Units do not serve the care of patients, but are intended for Funktion und Dysfunktion des Nervensystems bearbeitet. the diagnosis and for the scientific examination of • Clinical Research Units (CRU): Die Clinical Research patients, which are necessary within the framework of the Units dienen nicht der Krankenversorgung, sondern sind clinical cooperation projects. They are a distinctive feature für Diagnose und für wissenschaftliche Untersuchungen in the research structure of a university clinic and consti- von Patientinnen und Patienten gedacht, die im Rahmen tute the basis of the clinical research. der klinischen Kooperationsprojekte notwendig sind. Sie • Clinical Training Program (KAP): The basic idea of the sind eine Besonderheit in der Forschungsstruktur einer clinical training program is to make the transition into Universitätsklinik und sind die Basis für die klinische For- basic research at the MDC easy for doctors with comple- schung. ted clinical training through targeted fostering of junior • Klinisches Ausbildungsprogramm (KAP): Grundidee group leaders and to lay the foundation for further scien- des klinischen Ausbildungsprogramms ist es, durch ge- tific training. zielte Nachwuchsförderung Medizinerinnen und Medizi- nern mit abgeschlossener klinischer Ausbildung den Über- 175

The entire funding for research which the MDC provides for gang in die Grundlagenforschung am MDC zu erleichtern the joint projects (“Twinning Grants”) of clinicians and basic und den Grundstein für die wissenschaftliche Weiterbil- research scientists is monitored by internal and external dung zu legen. experts. The success of this model was evident through the positive results of an independent evaluation of the clinical Die gesamte Forschungsförderung, die das MDC für die ge- research groups in the Cardiovascular and Cancer Research meinsamen Projekte („Twinning Grants“) von Klinikern und Programs that took place in April 2002. As was seen in the Gruppen der Grundlagenforschung aufbietet, wird von inter- evaluation report of the program oriented funding, the value nen und externen Experten evaluiert. Der Erfolg dieses of the MDC’s interdisciplinary concept in the various fields Modells lässt sich an den positiven Evaluationsergebnissen of basic and clinical research was confirmed. Specifically, the der Begutachtung der klinischen Arbeitsgruppen im Bereich auditors highly rated the three structural foci (KKP, CRU, and der Herz-Kreislauf- sowie Krebsforschung im April 2002 KAP). belegen. Wie auch bei der Begutachtung im Rahmen der Pro- grammorientierten Förderung wurde das interdisziplinäre A New Partner, The HELIOS Clinics GmbH Konzept des MDC in den verschiedenen Bereichen der As of June 1, 2001, the HELIOS Kliniken GmbH Fulda Grundlagenforschung und der klinischen Anwendung voll became the financial provider of the Buch Clinic as well as bestätigt. Dabei haben die Gutachter die drei strukturellen the two Charité Clinics, the Robert Rössle Cancer Clinic and Schwerpunkte KKP, CRU und KAP insgesamt als besonders the Franz Volhard Cardiovascular Clinic. Thus, it is assured positiv bewertet. that patient care and research can continue in close cooperation with the Charité and private fund providers. The Ein neuer Partner, die HELIOS Kliniken GmbH HELIOS Kliniken GmbH has begun with the new construc- Zum 1. Juni 2001 hat die HELIOS Kliniken GmbH Fulda als tion of a clinic for approximately 200 million Euros in the di- Träger das Klinikum Buch sowie die beiden Berlin-Bucher rect vicinity of the Berlin-Buch Campus which will be com- Charité-Kliniken, Robert-Rössle-Krebsklinik und Franz-Vol- pleted at the end of 2005. The Buch University Clinics of the hard-Herz-Kreislaufklinik, übernommen. Künftig ist damit Charité, the FVK, and the RRK will move into the new sichergestellt, dass Forschung und Krankenversorgung in en- building together with the Buch Clinics Complex with a total ger Kooperation von Charité und privatem Träger erfolgen of 1,069 beds. Thus, one of the most modern and largest können. Die HELIOS Kliniken GmbH hat in unmittelbarer clinics of Berlin will be located in the direct vicinity of the Nähe des Campus Berlin-Buch mit dem Neubau einer Klinik Berlin-Buch Campus. With the founding of the Helios Re- für rund 200 Millionen Euro begonnen, der Ende 2005 fertig- search Center GmbH through a Shareholder Contract in June gestellt sein wird. In das neue Gebäude werden die Bucher 2002 a first formalized partnership with the private operator Universitätskliniken der Charité, zusammen mit dem Bucher HELIOS GmbH has been formed on the part of the MDC Klinikum mit insgesamt 1069 Betten einziehen. Damit wird Berlin-Buch. The task of the company is to support further sich eine der modernsten und größten Kliniken Berlins direkt science and research in the HELIOS group of teaching hospi- neben dem Campus Berlin-Buch befinden. Mit der Gründung tals, in particular in the sector of clinical studies, and to der Helios Research Center GmbH über einen Gesellschafter- obtain third party funding for their financing. vertrag im Juni 2002 ist eine erste formalisierte Partnerschaft zum privaten Betreiber HELIOS GmbH seitens des MDC Berlin-Buch hergestellt worden. Aufgabe der Gesellschaft ist es, Wissenschaft und Forschung in der HELIOS Kliniken Gruppe, insbesondere im Bereich klinischer Studien, zu unter- stützen und Drittmittel zu deren Finanzierung einzuwerben.

Die Robert-Rössle-Krebs-Klinik der Charité/Helios Klinikum Berlin auf dem Campus Das Experimental and Clinical Research Center Charité/ Berlin-Buch. Ärzte dieser Klinik arbeiten eng mit Wissenschaftlern des MDC Berlin-Buch MDC Berlin-Buch (ECRC) zusammen. Copyright: Helios Klinikum Berlin/Photograph: Thomas Oberländer Eine wichtige Schlussfolgerung aus den Begutachtungen des The Robert Roessle Cancer Clinic of the Charité/Helios Klinikum Berlin on the Berlin- MDC und den Kliniken in Berlin-Buch ist die Verstärkung Buch Campus. Physicians of this clinic work closely together with scientists of the MDC und der Ausbau von Schnittstellen und institutionellen Ver- Berlin-Buch. Copyright: Helios Klinikum Berlin/Photographer: Thomas Oberländer bindungsbrücken zwischen der Grundlagenforschung und der Klinik. Diese Überlegungen haben zum Konzept geführt, die klinischen Forschungsprojekte des MDC und der Kliniken zu einem „Experimental and Clinical Research Center“ Charité/ MDC Berlin-Buch (ECRC) zusammenzuführen. Es soll in der 2005/2006 frei werdenden Robert-Rössle-Klinik (Cha- rité) und in einem Gebäude in direkter Verbindung zum HELIOS-Klinikum Berlin-Buch angesiedelt werden. Die Verbindung des Potentials des Forschungsbereichs Gesund- heitsforschung der Helmholtz-Gemeinschaft mit der Charité- Universitätsmedizin Berlin und Partnern der Industrie wie Schering, Philips, General Electric und Siemens im Bereich der Entwicklung von NMR-Technologien bietet die Möglich- keit, solch ein Zentrum von nationaler und internationaler Bedeutung zu schaffen. 176

Die Franz-Volhard-Herz-Kreislauf-Klinik der Charité/Helios Klinikum Berlin auf dem Campus Berlin-Buch. Ärzte dieser Klinik arbeiten eng mit Wissenschaftlern des MDC Berlin-Buch zusammen. Copyright: Helios Klinikum Berlin/Photograph: Thomas Oberländer The Franz Volhard Clinic for Cardiovascular Diseases of the Charité/Helios Klinikum Berlin on the Berlin-Buch Campus. Physicians of this clinic work closely together with scientists of the MDC Berlin-Buch. Copyright: Helios Klinikum Berlin/Photographer: Thomas Oberländer

The Experimental and Clinical Research Center Charité/ Das MDC Berlin-Buch und die Helmholtz- MDC Berlin-Buch (ECRC) Gemeinschaft The expert examinations of the MDC and the clinics in Berlin-Buch confirmed the necessity of setting up interfaces Die Helmholtz-Gemeinschaft and institutional links between basic research and the clinic. Die Helmholtz-Gemeinschaft (HGF) ist mit ihren 15 For- These suggestions lead to the concept of combining the clini- schungszentren und einem Jahresbudget von rund 2,1 Milli- cal research projects of the MDC and the clinics in an “Exper- arden Euro die größte Wissenschaftsorganisation Deutsch- imental and Clinical Research Center” Charité/MDC Berlin- lands. Die 24.000 Mitarbeiterinnen und Mitarbeiter der Buch (ECRC). The ECRC will be located in the current Helmholtz-Gemeinschaft arbeiten in sechs Forschungsberei- Robert-Rössle-Clinic (Charité) building which will become chen. Zehn Helmholtz-Zentren arbeiten auf dem Gebiet der available in 2005/2006 and in a building directly linked to the Gesundheit. Die vier größten unter ihnen sind das GSF For- HELIOS Clinics Berlin-Buch. The linking of the potential of schungszentrum für Umwelt und Gesundheit GmbH, das the research sector Health Research of the Helmholtz Asso- DKFZ Krebsforschungszentrum Heidelberg, die GBF Gesell- ciation with the Charité University Medicine Berlin and part- schaft für Biotechnologische Forschung GmbH und das ners in industry, such as Schering, Philips, General Electric, MDC. Die Zentren der Gesundheitsforschung arbeiten in ver- and Siemens, in the sector of the development of NMR Tech- schiedenen Bereichen der medizinischen Wissenschaft, ins- nologies offers the possibility of creating a center of national besondere in der Erforschung der biologischen Grundlagen, and international significance. der klinischen Anwendung und der allgemeinen, die Gesund- heit fördernden Maßnahmen (Public Health Research).

The MDC Berlin-Buch and the Helmholtz Im September 2001 wurde die HGF reformiert und das neue Association Instrument der programmorientierten Förderung (PROF) ein- geführt. Ziel dieser Förderung ist die Bereitstellung der Res- The Helmholtz Association sourcen für die sechs Forschungsbereiche, indem thematisch The Helmholtz Association (HGF) is the largest scientific Programme definiert sind, die auf der Basis von Kooperation organization in Germany with its 15 research centers and an und Wettbewerb nach externer Evaluierung zu langfristigen annual budget of around 2.1 billion Euros. The 24,000 Forschungszielen durchgeführt werden. Die Helmholtz-Zen- employees of the Helmholtz Association work in six research tren erstellen im Verbund oder einzeln ihre Programme in sectors. Ten Helmholtz centers work in the field of health eigener Verantwortung und stellen sich mit diesen einem research. The four largest among them are the GSF Research Wettbewerb innerhalb der Helmholtz-Gemeinschaft. Die Pro- Center for the Environment and Health GmbH, the DKFZ gramme und Beiträge der einzelnen Zentren werden von inter- Cancer Research Center, Heidelberg, the GBF Society for nationalen Experten begutachtet. Die daraus resultierende Biotechnological Research GmbH, and the MDC. The health Förderempfehlung des HGF Senats ist Grundlage für die research centers work in various areas of medical science, in Finanzierung der Zentren durch die Zuwendungsgeber. Inner- 177

particular in the exploration of the basics of biology, clinical halb des Forschungsbereiches Gesundheit sind sieben Pro- applications, and general measures for the promotion of gramme definiert worden. Das MDC Berlin-Buch ist dabei an health (Public Health Research). drei Programmen beteiligt: Erforschung von Herz-Kreislauf- und Stoffwechselerkrankungen, Krebsforschung sowie die Er- In September 2001, the HGF was reorganized and the new forschung der Funktion und Dysfunktion des Nervensystems. instrument of program-oriented funding (PROF) was introduced. The aim of the program-oriented funding is the provi- Die Programmorientierte Förderung sion of resources for the six research sectors, in which pro- Im Zusammenhang mit der programmorientierten Förderung grams are defined as regards to subject matter, which are (PROF) der Helmholtz-Gemeinschaft sind die Programme implemented as long-term research objectives on the basis of Krebsforschung (im Juli 2002 in Heidelberg), Funktion und cooperation and competition according to external evalua- Dysfunktion des Nervensystems (im Juli 2002 in Jülich) und tion. The Helmholtz centers create their programs in associa- Herz-Kreislauf- und Stoffwechsel-Erkrankungen (im Sep- tion or individually and thus place themselves in competition tember 2002 in Berlin-Buch) von international besetzten within the Helmholtz Association. The programs and contri- Gutachtergremien evaluiert worden. Im Gegensatz zu den butions of the individual centers are examined by internation- Evaluationen der Jahre 1996 bis 1998, wo einzelne Arbeits- al experts. The resulting recommendation for funding by the gruppen begutachtet wurden, war die Begutachtung zur HGF Senate is the basis for the financing of the centers by the programmorientierten Förderung eine rein strategische Pro- grant providers. Seven programs have been defined within grammbegutachtung. Als Ergebnis wurden die Programm- the research sector health. The MDC Berlin-Buch is involved teile Krebs und Neurowissenschaften als wissenschaftlich in the following three research programs: Cardiovascular and herausragend bezeichnet. Auch im Herz-Kreislaufprogramm Metabolic Diseases, Cancer Research, and Function and wurde den MDC-Forschungsgruppen eine große Produk- Dysfunction of the Nervous System

Program-Oriented Funding Grundsteinlegung für das Labor für Medizinische Genomforschung auf dem Campus In connection with the program-orientated funding (PROF) Berlin-Buch am 24. Juni 2003. Bauherren sind das MDC Berlin-Buch und das For- of the Helmholtz Association, the research programs Cancer schungsinstitut für Molekulare Pharmakologie (FMP). Die Maurerkelle schwingen (v. l.) Prof. Walter Rosenthal (FMP), der Regierende Bürgermeister von Berlin, Klaus Wowereit, Research (July 2002 in Heidelberg), Function and Dys- der MDC-Kuratoriumsvorsitzende, Ministerialdirektor Reinhard Junker vom Bundesfor- function of the Nervous System (July 2002 in Jülich) and schungsminisiterium und Prof. Detlev Ganten, MDC-Stiftungsvorstand. Copyright: MDC/Photograph: Siegfried Röpke Cardiovascular and Metabolic Diseases (September 2002 in Laying the foundation stone for the Laboratory for Medical Genomics on the Berlin-Buch Berlin-Buch) were evaluated by expert committees with Campus on June 24, 2003. It is jointly erected by the MDC Berlin-Buch and the For- internationally drawn members. In contrast to the evaluations schungsinstitut für Molekulare Pharmakologie (FMP). Swinging the trowel are (from left): Prof. Walter Rosenthal (FMP), the Governing Mayor of Berlin, Klaus Wowereit, the chair- of the years 1996 to 1998, where individual laboratory groups man of MDC’s Board of Trustees, Ministerialdirektor Reinhard Junker from the Federal were examined, the examination in respect to PROF was a Research Ministry, and Detlev Ganten, M.D., Ph.D, MDC’s Scientific Director. Copyright: MDC/Photographer: Siegfried Röpke strategic program examination. It resulted in the program sections Cancer and Neurosciences being described as scien- tifically outstanding. In the Cardiovascular Program, great productivity was attested in the MDC research groups and their work was assessed as competitive and above the international average. This high estimation of the research work of the MDC Berlin-Buch was also confirmed overall by the ISI- Analysis in the National Citation Report 2001. According to this analysis, the MDC Berlin-Buch takes one of the top positions among the German Institutes conducting molecular medical research.

Laboratory for Medical Genome Research Already before the HGF evaluation occurred, the construction of a new building for medical genome research on the Berlin-Buch Campus, supported with considerable European funds (European Fund for Regional Development (EFRE)), had been confirmed within the framework of the program- oriented funding by the grant providers. In the opinion of the experts, the surplus funding received within the framework of program-oriented funding to expand the MDC Berlin-Buch in the area of medical genome research is justified.

The construction of the Laboratory for Medical Genome Research is a joint project of the MDC Berlin-Buch and the Institute for Molecular Pharmacology (FMP) and, thus, 62.5% of the costs will be financed by the EFRE. The new laboratory building will be constructed at the end of the main axis of the Campus grounds in the direct vicinity of the FMP 178

Der Campus Berlin-Buch, 2003 The Campus Berlin-Buch, 2003

and the Walter-Friedrich House of the MDC. The completion tivität attestiert und ihre Arbeit als kompetitiv und über dem is planned for the spring of 2005. The new building of the internationalen Durchschnitt bewertet. Diese hohe Einschät- Laboratory for Medical Genome Research will serve to create zung der Forschungsarbeiten des MDC Berlin-Buch wird the spatial preconditions necessary for investigating new auch insgesamt durch die Analyse des Institute for Scientific questions in genome research. Information (ISI) in Philadelphia/USA im National Citation Report 2001 bestätigt. Nach dieser Analyse nimmt das MDC Berlin-Buch unter den deutschen Instituten, die molekulare The MDC Berlin-Buch and the Campus Berlin-Buch medizinische Forschung betreiben, einen der vordersten Plätze ein. BBB Management GmbH with Innovation and Founder Center The BBB Management GmbH (BBB) was founded by the Labor für medizinische Genomforschung Max Delbrück Center for Molecular Medicine (MDC) Bereits vor dem Begutachtungsverfahren im Rahmen der pro- Berlin-Buch in 1995. The Forschungsverbund Berlin as well grammorientierten Förderung war vonseiten der Zuwen- as Schering AG are co-shareholders (20 percent each). The dungsgeber ein Neubau für die medizinische Genomfor- BBB acts as the development and coordination company for schung auf dem Campus Berlin-Buch mit erheblichen the entire research campus in Berlin-Buch, managing a com- europäischen Mitteln (EFRE) bestätigt worden. Die Voten der mercial area of around 26,000 square meters. As such, it ope- Gutachter zu den Überzeichnungen im Rahmen der pro- rates an Innovation and Founder Center (IGZ) for new bio- grammorientierten Förderung haben die Ausbauziele des technology companies and is in charge of the real estate on MDC Berlin-Buch im Bereich der medizinischen Genom- the Campus. Here, small and medium-sized enterprises carry forschung unterstützt. Das Labor für Medizinische Genom- out research and production. In 2003, several enterprises, in- forschung ist ein gemeinsamer Bau von MDC Berlin-Buch cluding three start-up companies, moved onto the Campus. und dem Forschungsinstitut für Molekulare Pharmakologie Currently, around 40 companies are located on the Campus of (FMP), dabei werden 62,5 Prozent der Kosten aus dem Euro- which around 30 are in the biotechnology sector. The compa- päischen Fonds für Regionalentwicklung (EFRE) finanziert. nies employ a total of around 500 employees. Der Laborneubau wird am Ende der Hauptachse des Campus- geländes in direkter Nachbarschaft zum FMP und dem Wal- In 2003, the BBB Management GmbH opened a Laboratory ter-Friedrich-Haus des MDC errichtet. Die Fertigstellung ist and Bio Computer Science building constructed at a cost of für Frühjahr 2005 vorgesehen. Mit dem Neubau des Labors around 16 million Euros and, simultaneously, celebrated the für Medizinische Genomforschung werden die räumlichen five-year existence of the IGZ. The new building has 8,000 Kapazitäten ausgebaut und damit die Voraussetzungen ge- square meters of commercial space and serves as the head- schaffen, neue Fragestellungen in der Genomforschung zu quarters for various enterprises. The extension of the IGZ has bearbeiten been completed with this new building. Therefore, the Berlin- Buch Campus has one of the largest sector-specific centers for new businesses in Germany at its disposal. Das MDC Berlin-Buch und der Campus-Berlin-Buch

Life Science Learning Laboratory BBB Management GmbH mit Innovations- und Gründer- In the Life Science Learning Lab (Gläsernes Labor) visitors zentrum can independently carry out gene technology and cell bio- Die BBB Management GmbH (BBB) wurde 1995 durch das logical experiments in authentic research laboratories and Max-Delbrück-Centrum für Molekulare Medizin (MDC) Ber- discuss their concrete applications in research, medicine, and lin-Buch gegründet. Mitgesellschafter sind der Forschungs- 179

biotechnology with scientists. Since 2001, the Life Science verbund Berlin (20 %) sowie die Schering AG (20 %). Die Learning Laboratory has participated in the “Long Night of BBB ist die Entwicklungs- und Betreibergesellschaft des Sciences” on the Berlin-Buch Campus with over 3,000 people Campus Berlin-Buch. Auf insgesamt ca. 26.000 m2 Nutz- per year visiting the Berlin-Buch campus. In 2003 alone, fläche stehen, insbesondere im geförderten Innovations- und more than 7,100 visitors took advantage of the advanced Gründerzentrum (IGZ), zu attraktiven Konditionen Gewerbe- training and continuing education classes including courses einheiten zur Verfügung, in den kleine und mittelständige in gene technology, cell biology, and protein analysis. In Unternehmen forschen und produzieren. Es gelang 2003, addition, in 2003 a new opportunity for laboratory workers to mehrere neue Unternehmen, darunter auch drei Neugründun- gain qualifications in molecular biology was offered jointly gen, auf dem Campus anzusiedeln. Gegenwärtig sind auf dem with the German authority for monitoring technical standards Campus rund 40 Unternehmen angesiedelt, davon sind circa (TÜV Academy Berlin). In 2003, the BBB Management 30 Biotechnologieunternehmen. Die Firmen beschäftigen GmbH opened an information bureau within the Life Science insgesamt circa 500 Mitarbeiter. Learning Laboratory building. It has a Berlin-Buch Campus exhibition and is the starting point for guided tours of the 2003 hat die BBB Management GmbH Campus Berlin-Buch Campus. ein mit rund 16 Millionen Euro errichtetes Labor- und Bio- informatikgebäude eröffnet und damit gleichzeitig das fünf- jährige Bestehen des IGZ gefeiert. Der Neubau hat eine ver- mietbare Fläche von rund 8.000 m2 und ist Sitz verschiedener Unternehmen. Mit dem Neubau wurde der Ausbau des IGZ abgeschlossen. Damit verfügt der Campus über eines der größten branchenspezifischen Gründerzentren in Deutschland.

Gläsernes Labor Im Gläsernen Labor können Besucher in authentischen For- schungslaboren gentechnische und zellbiologische Experi- mente durchführen und mit Wissenschaftlern über konkrete Anwendungen in Forschung, Medizin und Biotechnologie diskutieren. Seit dem Jahre 2001 konzipiert und realisiert die vom Gläsernen Labor der BBB GmbH koordinierte Öffent- lichkeitsarbeit zusammen mit allen Einrichtungen des Cam- pus die „Lange Nacht der Wissenschaften“ auf dem Campus Berlin-Buch. Über 3000 Besucher haben diese Veranstaltung pro Jahr auf dem Campus besucht. Allein im Jahre 2003 nutz-

Bundesjustizministerin Brigitte Zypries lässt sich von Dr. Ulrich Scheller im Gläsernen ten mehr als 7.100 Besucher die umfassenden Angebote zur Labor in die gentechnische Arbeitswelt einführen. (22. Juli 2003) Fort- und Weiterbildung inklusive Gentechnik, Zellbiologie Copyright: MDC/Photograph: Uwe Eising und Proteinenanalysen. In 2003 wurde gemeinsam mit der Introduction into the world of genetic engineering - the Federal Minister/Secretary of Justice, Brigitte Zypries and Dr. Ulrich Scheller in the Life Science Learning Lab. (July, TÜV Akademie Berlin ein neues Qualifizierungsangebot für 22, 2003) Laborkräfte angeboten – ein zehnwöchiger zertifizierter Wei- Copyright: MDC/Photographer: Uwe Eising terbildungskurs zur Fachkraft für Molekularbiologie. Im Herbst 2003 eröffnete die BBB Management GmbH in den Räumen des Gläsernen Labors ein Informationsbüro, das gleichzeitig Besuchertreff ist. Es zeigt eine Campusausstel- lung, und ist Ausgangspunkt für Campus-Führungen.

InnoRegio-Initiative Berlin-Buch InnoRegio Berlin-Buch InnoRegio is a funding program that was established in 1999 InnoRegio wurde 1999 vom Bundesministerium für Bildung by the German Federal Ministry of Research (BMBF). The und Forschung (BMBF) gestartet. Ziel des InnoRegio-Pro- InnoRegio program aims to encourage and support develop- gramms ist die Verstärkung von Entwicklung und Investition ment and investment in areas of science, education, and in den Bereichen Wissenschaft, Bildung und Wirtschaft, um business in order to make the specific region or location more Wertschöpfung und Wettbewerbsfähigkeit in bestimmten Re- commercially attractive and competitive. gionen zu steigern.

The InnoRegio Project has given rise to a clinical research Im Bereich klinische Forschung und Entwicklung ist mit dem network in Buch. The main focus of the initiative is to inter- InnoRegio-Projekt ein Bucher Netzwerk entstanden. Kernan- link the Berlin-Buch research institutes, clinics, and compa- liegen ist dabei die Vernetzung der in Berlin-Buch ansässigen nies in regard to developing novel forms of therapeutic ap- Forschungsinstitute, Kliniken und Unternehmen im Hinblick proaches, biomedical technologies, and clinical applications. auf die Entwicklung neuartiger therapeutischer Ansätze, bio- The first projects involve the fields of molecular tumor medizinischer Technologien und klinischer Anwendungen. diagnosis, genetic epidemiology based on twin studies, the Erste Projekte umfassen die Bereiche der molekularen preclinical and clinical testing and development of a new Tumordiagnostik, der genetischen Epidemiologie auf der lipid-based administration form (drug delivery system) for Basis von Zwillingsstudien, die präklinische und klinische the anticancer drug, Taxol, as well as protein- and active Prüfung und Entwicklung von neuen, lipidbasierten Darrei- 180

ingredient-screening. The funding for the InnoRegio Berlin- Buch through 2005 amounts to 5.2 million Euro.

Institute for Molecular Pharmacology (FMP) The Institute for Molecular Pharmacology (FMP) is engaged in basic research in the identification and characterization of biological macromolecules as drug targets. Through the close spatial proximity to the MDC, the already existing collaboration between the two research centers has been considerably intensified. The research concepts of the MDC and the FMP complement each other: while the molecular medical research at the MDC is particularly dedicated to diseases or clinical symptoms and their molecular explanations, the FMP investigates the functional and structural characterization of proteins as well as the development of strategies for their pharmacological influence.

The close connection between the two research establishments extends into the organizational level. Thus, large equipment is shared and jointly operated. Guest scientist contracts make it possible for scientists of one institute to use the Das Forschungsinstitut für Molekulare Pharmakologie (FMP) auf dem Campus Berlin- equipment in the other. Both establishments send representa- Buch, ein enger Kooperationspartner des MDC Berlin-Buch. Copyright: Helios Klinikum Berlin/Photograph: Thomas Oberländer tives to important committees of the other establishment, The Institute for Molecular Pharmacology (FMP) at the Berlin-Buch Campus, a close respectively. The planning of costly and long-term research collaborator of the MDC-Berlin-Buch. projects as well as the appointment of leading scientists takes Copyright: Helios Klinikum Berlin/Photographer: Thomas Oberländer place in joint agreement. The MDC and the FMP arrange and finance joint events for those studying for their doctorates.

chungsform (drug delivery system) für Krebsmedikamente Taxol, sowie das Protein- und Wirkstoffscreening. Insgesamt wird InnoRegio Berlin-Buch bis zum Jahr 2005 mit 5,2 Mil- lionen Euro gefördert.

Forschungsinstitut für Molekulare Pharmakologie (FMP) Das Forschungsinstitut für Molekulare Pharmakologie (FMP) betreibt Grundlagenforschung zur Identifizierung und Nutz- barmachung potentieller Zielstrukturen für Pharmaka. Durch die räumliche Nähe wurde die bereits bestehende Zusam- menarbeit zwischen MDC und FMP erheblich intensiviert. Die Forschungskonzepte des MDC und des FMP ergänzen sich: Während sich die molekularmedizinische Forschung am MDC besonders Erkrankungen oder klinischen Symptomen und deren molekularen Erklärungen widmet, hat das FMP die funktionelle und strukturelle Charakterisierung von Protei- nen zum Ziel, sowie die Entwicklung von Strategien zu ihrer pharmakologischen Beeinflussung.

Auch auf der organisatorischen Ebene gibt es eine Verflech- tung beider Forschungseinrichtungen. So werden Großgeräte gemeinsam genutzt und gemeinsam betrieben. Gastwissen- schaftlerverträge ermöglichen es Wissenschaftlern der einen Einrichtung die Ausstattung einer gastgebenden Arbeits- gruppe in der anderen Einrichtung zu nutzen. Beide Institute entsenden Vertreter in wichtige Gremien der jeweils anderen Einrichtung. Die Planung aufwendiger und langfristiger For- schungsprojekte sowie die Berufung von leitenden Wissen- schaftlern erfolgen in gegenseitiger Absprache. Darüber hinaus finanzieren und gestalten MDC und FMP gemeinsam Veranstaltungen für Doktoranden. 181

Organizational Structure Organisationsstruktur

The Max Delbrück Center for Molecular Medicine (MDC) Das Max-Delbrück-Centrum für Molekulare Medizin (MDC) Berlin-Buch is a foundation under public law of the State of Berlin-Buch ist eine Stiftung des öffentlichen Rechts des Berlin with the purpose of pursuing medical research at the Landes Berlin mit dem Zweck, medizinische Forschung auf molecular and cellular levels and implementing its clinical molekularer und zellulärer Ebene und ihre klinische Anwen- application. As laid down in the bylaws, the bodies of the dung und Umsetzung zu betreiben. Wie im Stiftungsgesetz Foundation are comprised of the following two boards: des MDC verankert, sind die Organe der Stiftung: • The Board of Trustees • das Kuratorium • The Executive Board of the Foundation • der Stiftungsvorstand

The Board of Trustees Das Kuratorium

The Board of Trustees is the Supervisory Committee of the Das Kuratorium ist das Aufsichtsgremium des MDC und MDC and monitors the legality, appropriateness, and econo- überwacht die Rechtmäßigkeit, Zweckmäßigkeit und Wirt- mic viability of the management of the Foundation’s busi- schaftlichkeit der Führung des Stiftungsgeschäftes. Es ent- ness. It decides on the general objectives of the research and scheidet über die allgemeinen Forschungsziele und wichtigen important research policy and financial matters. Up until Sep- forschungspolitischen und finanziellen Angelegenheiten der tember 2002, Wolf-Michael Catenhusen, parliamentary Per- Stiftung. Bis September 2002 hatte der Parlamentarische manent Secretary of the Federal Ministry for Education and Staatssekretär Wolf-Michael Catenhusen vom Bundesminis- Research (BMBF), held the office. His successor as Chairman terium für Bildung und Forschung (BMBF) das Amt inne. is Reinhard Junker, Head of the Department of Health, Bio- Sein Nachfolger als Vorsitzender ist Ministerialdirektor Rein- sciences and Continuity in the BMBF. In 2004, a new ap- hard Junker, Abteilungsleiter Gesundheit, Biowissenschaf- pointment of the Vice Chairman will be announced following ten, Nachhaltigkeit im BMBF. Der stellvertretende Vorsitz the departure of Dr. Peer Pasternack, Permanent Secretary of wird nach Ausscheiden von Staatssekretär Dr. Peer Paster- the Berliner Senate Administration for Science, Research, nack von der Berliner Senatsverwaltung für Wissenschaft, and Culture. Forschung und Kultur im Jahre 2004 berufen.

Members of the Board of Trustees Mitglieder des Kuratoriums Head of Dept. Reinhard Junker, Federal Ministry for Educa- MinDir Reinhard Junker, Bundesministerium für Bildung tion and Research Berlin (Chairman) und Forschung, Berlin (Vorsitz) (To be named), Senate’s Administration for Science, Research N.N., Senatsverwaltung für Wissenschaft, Forschung und and Culture, Berlin (Vice Chairperson) Kultur, Berlin (stellv. Vorsitz) Prof. Günter Breithardt, Münster University Prof. Günter Breithardt, Universität Münster Prof. Hans R. Brunner, Centre Hospitalier Universitaire Prof. Hans R. Brunner, Centre Hospitalier Universitaire Vaudois, Lausanne/Switzerland Vaudois, Lausanne/Schweiz Prof. Manfred Dietel, Charité University Medicine, Berlin Prof. Manfred Dietel, Charité-Universitätsmedizin Berlin Prof. Bärbel Friedrich, Humboldt University, Berlin Prof. Bärbel Friedrich, Humboldt-Universität zu Berlin Prof. Falko Herrmann, Greifswald University Prof. Falko Herrmann, Universität Greifswald Prof. Dr. Reinhard Jahn, Max Planck Institute for Biophysical Prof. Dr. Reinhard Jahn, Max-Planck-Institut für biophysika- Chemistry, Göttingen lische Chemie, Göttingen 182

Das Max-Delbrück-Haus des MDC. Copyright: MDC/Photograph: Uwe Eising MDC’s Max Delbrück House Copyright: MDC/Photographer: Uwe Eising

Department Head Oskar-Peter Kaye, Federal Ministry for Ministerialrat Oskar-Peter Kaye, Bundesministerium für Bil- Education and Research Bonn dung und Forschung, Bonn Senator Dr. Heidi Knake-Werner, Senate’s Administration for Senatorin Dr. Heidi Knake-Werner, Senatsverwaltung für Health, Social Matters and Consumer Protection, Berlin Gesundheit, Soziales und Verbraucherschutz, Berlin Permanent Secretary Dr. Hermann Schulte-Sasse, of the Staatssekretär Dr. Hermann Schulte-Sasse, Senatsverwaltung Senate’s Administration for Health, Social Matters and Con- für Gesundheit, Soziales und Verbraucherschutz, Berlin sumer Protection, Berlin Prof. Dr. Dieter Lenzen, Freie Universität Berlin Prof. Dr. Dieter Lenzen, Free University of Berlin Dr. Gary R. Lewin, MDC, Berlin-Buch Dr. Gary R. Lewin, MDC, Berlin-Buch Prof. Jürgen Mlynek, Humboldt-Universität zu Berlin Prof. Jürgen Mlynek, Humboldt University, Berlin Prof. Annemarie Poustka, Deutsches Krebsforschungszen- Prof. Annemarie Poustka, German Cancer Research Center trum (DKFZ), Heidelberg (DKFZ), Heidelberg Regierungsdirektor Markus Siebels, Bundesministerium für Senior Government Officer Markus Siebels, Federal Ministry Finanzen, Berlin of Finance, Berlin Dr. Thomas Sommer, MDC, Berlin-Buch Dr. Thomas Sommer, MDC, Berlin-Buch Dr. Albert Statz, Bundesministerium für Gesundheit, Bonn Dr. Albert Statz, Federal Ministry for Health, Bonn Prof. Dr. Axel Ullrich, Max-Planck-Institut für Biochemie, Prof. Dr. Axel Ullrich, Max Planck Institute for Biochemistry, Martinsried Martinsried Prof. Dr. Kurt von Figura, Universität Göttingen Prof. Dr. Kurt von Figura, Göttingen University

The Scientific Committee Der Wissenschaftliche Ausschuss

The Scientific Committee of the Board of Trustees was Den Wissenschaftlichen Ausschuss des Kuratoriums leitete directed by Prof. Volker ter Meulen until 2003. The Scientific bis 2003 Prof. Volker ter Meulen. Der Wissenschaftliche Aus- Committee prepares the decisions of the Board of Trustees in schuss bereitet die Entscheidung des Kuratoriums in wissen- scientific questions. It bears the responsibility for the conti- schaftlichen Fragen vor. Er trägt die Verantwortung für die nual evaluation of results of the MDC’s research work fortlaufende Ergebnisbewertung der Forschungsarbeiten des through scientific examination. In addition to the scientific MDC durch wissenschaftliche Begutachtung. Dem Wissen- members of the Board of Trustees, external scientific specia- schaftlichen Ausschuss gehören neben den wissenschaft- lists are also members of the Scientific Committee. lichen Mitgliedern des Kuratoriums auch externe Wissen- schaftler an. 183

Members of the Scientific Committee Mitglieder des Wissenschaftlichen Ausschusses Prof. Dr. Kurt von Figura, Göttingen University Prof. Dr. Kurt von Figura, Universität Göttingen Prof. Rudi Balling, Society for Biotechnological Research Prof. Rudi Balling, Gesellschaft für Biotechnologische For- (GBF), Braunschweig schung (GBF), Braunschweig Prof. Günter Breithardt, Münster University Prof. Günter Breithardt, Universität Münster Prof. Hans R. Brunner, Centre Hospitalier Universitaire Vau- Prof. Hans R. Brunner, Centre Hospitalier Universitaire Vau- dois, Lausanne/Switzerland dois, Lausanne/Schweiz Prof. Bärbel Friedrich, Humboldt University Berlin Prof. Bärbel Friedrich, Humboldt-Universtität zu Berlin Prof. Dr. Roger Goody, Max Planck Institute for Molecular Prof. Dr. Roger Goody, Max-Planck-Institut für molekulare Physiology, Dortmund Physiologie, Dortmund Prof. Falko Herrmann, Greifswald University Prof. Falko Herrmann, Universität Greifswald Prof. Dr. Christoph Huber, Mainz University Prof. Dr. Christoph Huber, Universität Mainz Prof. Dr. Wieland Huttner, Max Planck Institute for Molecu- Prof. Dr. Wieland Huttner, Max-Planck-Institut für molekular Cell Biology and Genetics, Dresden lare Zellbiologie und Genetik, Dresden Prof. Dr. Reinhard Jahn, Max Planck Institute for Biophysical Prof. Dr. Reinhard Jahn, Max-Planck-Institut für biophysika- Chemistry, Göttingen lische Chemie, Göttingen Prof. Thomas Meitinger, GSF Research Center for the Envi- Prof. Thomas Meitinger, GSF Forschungszentrum für Um- ronment and Health, Neuherberg welt und Gesundheit, Neuherberg Prof. Dr. Leena Peltonen, Helsinki University, Finland Prof. Dr. Leena Peltonen, Universität Helsinki, Finnland Prof. Annemarie Poustka, German Cancer Research Center, Prof. Annemarie Poustka, Deutsches Krebsforschungszen- Heidelberg trum (DKFZ), Heidelberg Professor Sir George K. Radda, Oxford University, Great Professor Sir George K. Radda, Universität Oxford, Großbri- Britain tannien Prof. Dr. Axel Ullrich, Max Planck Institute for Biochemistry, Prof. Dr. Axel Ullrich, Max-Planck-Institut für Biochemie, Martinsried Martinsried

The Executive Board of the Foundation

The Executive Board of the Foundation directs the MDC and consists of a Scientific Director, his deputy, and an Admini- strative Director. Since January 1992, Professor Detlev Der Campus Berlin-Buch Ganten has been the Scientific Director of the MDC. His de- Copyright: MDC The Campus Berlin-Buch puty is Professor Walter Birchmeier. Dr. Waltraud Kreutz- Copyright: MDC Gers took over the administrative direction of the Institute until February 2003. Her successor was Dr. Stefan Schwartze in the summer of 2003.

The Scientific Council

Under the direction of Dr. Martin Lipp, the Scientific Council advises the Executive Board of the Foundation in matters of fundamental scientific importance. In addition to the MDC scientific program coordinators, additional MDC scientific staff members are members of the Scientific Council.

Members of the Scientific Council Prof. Walter Birchmeier Prof. Thomas Blankenstein Dr. Kurt Bommert Dr. Iduna Fichtner Dr. Hannelore Haase Der Stiftungsvorstand Prof. Udo Heinemann Dr. Uta Höpken Der Stiftungsvorstand leitet das Institut und besteht aus ei- Prof. Helmut Kettenmann nem wissenschaftlichen Mitglied und einem administrativen Prof. Dr. Gary Lewin Mitglied. Seit Januar 1992 ist Prof. Detlev Ganten wissen- Dr. Martin Lipp (Chairman) schaftlicher Direktor des MDC. Er wird vertreten durch Prof. Prof. Friedrich Luft Walter Birchmeier. Bis Februar 2003 hatte Dr. Waltraud Dr. Margret Irmgardt Moré Kreutz-Gers die administrative Leitung des Instituts über- Dr. Thomas Müller nommen. Ihre Nachfolge trat Dr. Stefan Schwartze im Som- Dr. Claus Scheidereit mer 2003 an. 184

Prof. Peter Schlag Der Wissenschaftliche Rat Dr. Katrin Stade Dr. Ruth Schmidt-Ullrich Der Wissenschaftliche Rat berät unter Leitung von Dr. Mar- Prof. Wolfgang Uckert (Vice Chairman) tin Lipp den Stiftungsvorstand in den Angelegenheiten von Dr. Gerd Wallukat grundsätzlicher wissenschaftlicher Bedeutung. Dem Wissen- schaftlichen Rat gehören neben den Koordinatoren der For- schungsschwerpunkte weitere wissenschaftliche Mitarbeiter The Staff Council des MDC an.

The Staff Council is primarily involved with decisions at the Mitglieder des Wissenschaftlichen Rates MDC which concern personnel issues. The chairperson of the Prof. Walter Birchmeier Staff Council is Mrs. Marion Bimmler. Prof. Thomas Blankenstein Dr. Kurt Bommert Members of the Staff Council Dr. Iduna Fichtner Marion Bimmler (Chairperson) Dr. Hannelore Haase Gabriele Born Prof. Udo Heinemann Lutz Else (Vice Chairperson) Dr. Uta Höpken Ingrid Grunewald Prof. Helmut Kettenmann Frank-Peter Kirsch Prof. Dr. Gary Lewin Ilse-Maria Knoblich Dr. Martin Lipp (Vorsitz) Dr. Peter Konzer Prof. Friedrich Luft Bernd Lemke (Vice Chairperson) Dr. Margret Irmgardt Moré Jana Richter Dr. Thomas Müller Christel Westen Dr. Claus Scheidereit Dr. Reinhard Zeisig Prof. Peter Schlag Dr. Katrin Stade Dr. Ruth Schmidt-Ullrich Prof. Wolfgang Uckert (stellv. Vorsitz) Dr. Gerd Wallukat

Der Personalrat

Der Personalrat ist insbesondere an solchen Entscheidungen des MDC Berlin-Buch beteiligt, welche die personellen und sozialen Belange der Beschäftigten betreffen. Personalrats- vorsitzende ist Frau Marion Bimmler.

Mitglieder des Personalrates Marion Bimmler (Vorsitz) Gabriele Born Lutz Else (stellv. Vorsitz) Ingrid Grunewald Frank-Peter Kirsch Ilse-Maria Knoblich Dr. Peter Konzer Bernd Lemke (stellv. Vorsitz) Jana Richter Christel Westen Dr. Reinhard Zeisig 185

Supporting Divisions Stabsstellen

Safety Sicherheit

The task of the MDC Safety Group is to ensure the safety of Aufgabe der Gruppe Sicherheit ist es, die Mitarbeiter des the MDC employees during their working hours and to pre- MDC vor Gefährdungen bei ihren unterschiedlichen Tätig- vent occupational accidents and work-related diseases. One keiten zu schützen, d.h. Arbeitsunfälle und berufsbedingte of the most important functions of the safety officers is risk Erkrankungen zu verhindern. Eine der wichtigsten Aufgaben assessment, i.e. the identification and assessment of risks dur- der Sicherheitsfachkräfte besteht in der Gefährdungsbeurtei- ing the handling of hazardous substances and biological lung, d.h. in der Identifizierung und Beurteilung von Risiken agents. The Occupational Safety Committee at the MDC con- während des Umgangs mit gefährlichen Stoffen und biologi- sists of the radiation protection officer, the biosafety officer, schen Agenzien. Der Arbeitsschutzausschuss am MDC setzt the employee physician, a representative of the Staff Council, sich zusammen aus dem Strahlenschutzbevollmächtigten, der and a safety representative from each laboratory and is direct- Beauftragten für Biologische Sicherheit, der Betriebsärztin, ed by the Administrative Director. einem Vertreter des Personalrates, einem Sicherheitsbeauf- tragten aus jeder Arbeitsgruppe und wird vom Administra- Head: Dr. Regina Möhring tiven Vorstand geleitet.

Leiterin: Dr. Regina Möhring

Hermann-von-Helmholtz-Haus Copyright: MDC, Photographer: Uwe Eising 186

Building and Technology Bau und Technik

Façade Fassade The extensive overall renovation measures at the Max- Die umfangreichen Gesamtsanierungsmaßnahmen am Max- Delbrück House were completed in the first half of 2002 with Delbrück-Haus sind in der ersten Hälfte des Jahres 2002 mit the renovation of the façade and the roof. The building shell der Sanierung der Fassade und des Daches abgeschlossen was renewed corresponding to the current requirements worden. Die Gebäudehülle wurde entsprechend der heutigen regarding protection against heat loss, laboratory operations, Anforderungen an Wärmeschutz, Laborbetrieb und notwen- and necessary maintenance. diger Instandhaltung erneuert.

Combined Office and Animal Experiment Building Kombiniertes Tierlabor- und Theoriegebäude The construction measure “New construction of an Animal Die Baumaßnahme „Neuerrichtung eines Tierlabor- und Laboratory and Office Building” for joint use by the MDC Theoriegebäudes“ für die gemeinsame Nutzung durch das and the Institute for Molecular Pharmacology (FMP) was MDC und das Forschungsinstitut für Molekulare Pharma- authorized in the year 2000. The five-story new building kologie (FMP) wurde im Jahr 2000 genehmigt. Der fünf- (Hermann-von-Helmholtz-House) consists of a unit for ani- stöckige Neubau (Hermann-von-Helmholtz-Haus) besteht mal experiments and an office unit. The administration and aus einer tierexperimentellen und aus einer theoretischen some research groups of the MDC moved into this building in Einheit. Es wurde im Frühjahr 2003 von der Verwaltung und the spring of 2003. The Animal Experiment Building will be einigen Forschungsgruppen des MDCs bezogen. Das tierex- completed in May 2004 and be occupied by the end of the perimentelle Gebäude wird im Mai 2004 baulich fertigge- year 2004. stellt und bis Ende des Jahres 2004 bezogen sein.

Laboratory for Medical Genome Research Labor für Medizinische Genomforschung The MDC and FMP are establishing a joint Laboratory build- Das MDC und FMP errichten gemeinsam ein Laborgebäude ing for the Medical Genome Research. The construction work für die Medizinische Genomforschung. Die Bauarbeiten wur- commenced at the end of 2003 and completion is scheduled den Ende 2003 begonnen, und werden voraussichtlich pla- for 2005. nungsgemäß im Jahr 2005 abgeschlossen.

Head: Grit Kuhlmann Leiterin: Grit Kuhlmann

Das Max Delbrück Communications Center (MDC.C) (li.) und das Hermann-von-Helm- Revision und Recht – Patente und Lizenzen holtz-Haus des MDC. Letzteres war am 6. Mai 2003 eingeweiht worden. Copyright: Helios Klinikum Berlin/Photograph: Thomas Oberländer The Max Delbrück Communications Center (MDC.C) (left) and the Hermann-von-Helm- Innenrevision holtz-House of the MDC, the latter having been inaugurated on May 6, 2003. Copyright: Helios Klinikum Berlin/Photographer: Thomas Oberländer Aufgabe der Revision ist es zu überprüfen, ob Gesetze, Ver- ordnungen, Richtlinien und Weisungen des Stiftungsvorstan- des eingehalten und ob die zugewiesenen öffentlichen Mittel zweckentsprechend, sparsam und wirtschaftlich eingesetzt werden. Dazu werden aufgrund eines vom Vorstand bestä- tigten jährlichen Revisionsplanes Ordnungsmäßigkeits- und Organisationsprüfungen vorgenommen. Zur Aufgabe der In- nenrevision gehört auch die Zusammenarbeit mit externen Prüfern und die Erarbeitung von Stellungnahmen zu den Berichten dieser externen Prüfer.

Rechtsangelegenheiten Schwerpunkt der Arbeit in diesem Bereich ist die Vorberei- tung und der Abschluss von Forschungsverträgen mit Indu- striepartnern und Kooperationsverträgen mit anderen For- schungseinrichtungen.

Patente und Lizenzen In diesem Bereich geht es vor allem darum, als unmittelbarer Ansprechpartner für alle Wissenschaftlerinnen und Wissen- schaftler in Fragen des Technologietransfers zur Verfügung zu stehen. Hier erfolgt eine enge Zusammenarbeit mit der aus der Life-Science-Stiftung hervorgegangenen Verwertungs- agentur Ascenion GmbH.

Leiterin: Anja Frahn 187

Auditing, Legal Affairs, Patents, and Licenses

Internal Audit The goal of the internal audit is to examine whether laws, official regulations, guidelines, and instructions from the Foundation Board of the MDC are being observed and whether the allocated public funds are being used appropriately, sparingly, and economically. For this reason, audits of the compliance with regulations and the organization are undertaken on the basis of an annual audit plan confirmed by the Foundation Board. The collaboration with external auditors and the drawing up of statements in respect to the reports of these external auditors is also included in the task of the internal audit.

Legal Matters The main focus of the work in this sector is the preparation and finalizing of research contracts with partners from industry and cooperation contracts with other research establishments.

Patents and Licenses This sector involves serving as the direct contact for all scientists in questions of technology transfer. Here, we maintain a close collaboration with the intellectual property management company Ascenion GmbH, which emerged from the „L’Homme“, Bronze, 1963, Jean Ipoustéguy Life Science Trust. Copyright: MDC, Photographer: Gunter Lepkowski

Head: Anja Frahn

Technology Transfer Technologietransfer

In the years 2002 and 2003, the sector of technology transfer In den Jahren 2002 und 2003 hat das MDC den Bereich Tech- at the MDC underwent an extensive re-organization. An nologietransfer einer umfassenden Neustrukturierung unter- external partner, Ascenion GmbH, began managing the eva- zogen. Mit der Be- und Verwertung der am MDC entwickel- luation and exploitation of the technologies and industrial ten Technologien und Schutzrechte wurde ein externer property rights developed at the MDC. Partner, die Ascenion GmbH, beauftragt.

Inventions and Existing Patent Portfolio of the MDC Erfindungen und bestehendes Patentportfolio des MDC Inventions from MDC employees are evaluated by Ascenion Erfindungen von MDC-Mitarbeitern werden von der Asce- GmbH with regards to patents and all issues relevant to the nion GmbH hinsichtlich patent- und vor allem marktrelevan- market. In the years 2002 and 2003, patents were applied for ter Gesichtspunkte bewertet. In den Jahren 2002 und 2003 in respect to 21 inventions from the MDC. The MDC patent wurden 21 Erfindungen vom MDC zum Patent angemeldet. portfolio comprises approximately 85 patent families. It Das MDC-Patentportfolio umfasst etwa 85 Patentfamilien. consists of industrial property rights, quality to the extent of Es besteht aus Schutzrechten, die einer eingehenden Begut- protection to be achieved, reachable markets, and the achtung unter anderem hinsichtlich ihres Patentierungsver- prospects for exploitation. The portfolio is characterized by laufs, der Qualität des zu erlangenden Schutzumfanges, des high innovation earnings that are available to the MDC now. erreichbaren Marktes und der Verwertungsaussichten unter- zogen wurden. Commercial Exploitation of Technologies The commercialization of scientific results has taken on an Kommerzielle Verwertung von Technologien increasing importance for the MDC and its employees in Die Kommerzialisierung wissenschaftlicher Ergebnisse hat recent years. Thus, it was possible to increase the income für das MDC und seine Mitarbeiter in den letzten Jahren from license and option contracts by more than 250,000 o. einen immer wichtigeren Stellenwert eingenommen. So Additional income of over 190,000 o has been generated konnten die Einnahmen aus Lizenz- und Optionsverträgen during the report period from agreements on transfer of um mehr als 250.000 o gesteigert werden. Aus Überlas- tangible properties in respect to animal models. sungsverträgen für Tiermodelle wurden in diesem Zeitraum zusätzlich Einnahmen in Höhe von über 190.000 o generiert. Contact: Karen Uhlmann, Ascenion GmbH Kontakt: Karen Uhlmann, Ascenion GmbH 188

Press and Public Relations Presse- und Öffentlichkeitsarbeit

The attention of the media and the public has been increas- Das Augenmerk der Medien und der Öffentlichkeit richtet ingly directed on biomedicine and gene research and, thus, sich zunehmend auf die Biomedizin und damit immer stärker ever increasingly on the Max Delbrück Center for Molecular auch auf das Max-Delbrück-Centrum für Molekulare Medi- Medicine (MDC) Berlin-Buch. Primarily supported by zin (MDC) Berlin-Buch. Das MDC wird mit öffentlichen federal taxpayer contributions, the MDC strives to communi- Geldern finanziert. Es ist deshalb bemüht, der Öffentlichkeit cate its work and progress to a broad public. To facilitate this und damit dem Steuerzahler in verständlicher Form zu ver- communication, the MDC Press Office initiates the following mitteln, für welche Forschungen es diese Mittel einsetzt. activities. 124 Pressemitteilungen in Deutsch und Englisch recherchier- The MDC Press Office published 124 press releases in te und verfasste die MDC-Pressestelle 2002/2003 und zeich- German and English in 2002/2003. It was also responsible for nete auch verantwortlich für die Pressearbeit für mehrere the promotion of several international congresses, such as internationale Kongresse. Dazu gehören auch Pressekon- one for the 2002 recipient of the Nobel Prize for Chemistry, ferenzen, die die MDC-Pressestelle inhaltlich und organi- Prof. Kurt Wüthrich (Swiss Federal Institute of Technology satorisch leitete, u.a. 2002 mit dem frisch gekürten Chemie- (ETH) Zurich, Switzerland). Within the framework of the nobelpreisträger Prof. Kurt Wüthrich (Eidgenössische European Initiative for Communicators in Science (EICOS) Technische Hochschule, ETH, Zürich/Schweiz). Im Rahmen for 2002 and 2003, the MDC Press Office organized and der European Initiative for Communicators in Science hosted the respective one-week visits of four journalists. In (EICOS) bereitete die MDC-Pressestelle für 2002/2003 in- addition, in September 2003, the MDC Press Office prepared haltlich und organisatorisch die jeweils einwöchigen Besuche the visit of a group of ten Chinese journalists who were visit- von vier Journalisten aus dem In- und Ausland vor und being Germany at the invitation of the German Federal Govern- treute sie vor Ort. Weiter bereitete sie im September 2003 den ment. Besuch einer Gruppe von zehn chinesischen Journalisten vor, die auf Einladung der Bundesregierung eine Informations- During 2002/2003, the MDC Press Office generated over reise durch Deutschland machten, und betreute sie zusammen 6,000 newspaper articles, which corresponds to a circulation mit MDC-Wissenschaftlern. of more than 600 million. The topics conveyed to the media were broadcast by over 20 television stations in both regional Die MDC-Pressearbeit war 2002/2003 Anlass für über 6.000 as well as national transmissions. This included contributions Zeitungsartikel, die einer Auflage von mehr als 600 Millio- to the German foreign broadcaster Deutsche Welle, to the nen entsprechen, in denen über die Forschungsarbeiten von Franco-German channel ARTE, to American Public Televi- MDC, und gemeinsamen Projekten von MDC, Kliniken und sion, to the BBC (Europe Direct), as well as to the ZDF Biotechfirmen berichtet wurde. MDC-Themen wurden in [Second German Channel], which, in its three-part series on über 20 Fernsehbeiträgen regionaler und überregionaler Sen- “Gene Hunters”, also reported on the German-Canadian re- der aufgegriffen, darunter von der Deutschen Welle, dem search project of Prof. Ludwig Thierfelder on the cardio- deutsch-französischen Kanal ARTE, dem amerikanischen vascular research of the MDC/Franz-Volhard Clinic. In addi- Public Television, der BBC (Europe Direct) sowie des ZDF, tion, over 30 radio contributions about the MDC were das in seiner dreiteiligen Serie über „Genjäger“ auch über ein broadcast by public radio stations. Furthermore, online news deutsch-kanadisches Forschungsprojekt von Prof. Ludwig services acquired a significant amount of information from Thierfelder (MDC/Franz-Volhard-Klinik) berichtete. Weiter the MDC Press Office via the Internet. The MDC Press wurden über 30 Hörfunkbeiträge über das MDC gesendet. In Office arranged around 500 Interviews for daily newspapers, großem Umfang griffen auch Online-Dienste im Internet 189

Aufmerksam verfolgen die beiden Kinder ein Experiment im „Chemietruck Justus“ des Bundesforschungsministeriums, der im Rahmen der Langen Nacht der Wissenschaften am 14. Juni 2003 auf den Campus Berlin-Buch gekommen war. Copyright: Helios Klinikum Berlin/Photograph: Thomas Oberländer Very concentrated watching an experiment on the “Chemistry truck Justus” of the Federal Research Ministry, which had come to the Berlin-Buch Campus on June 14, 2003. Copyright: Helios Klinikum Berlin/Photographer: Thomas Oberländer

magazines, weekly newspapers, television, and radio as well Informationen der MDC-Pressestelle auf. Im Berichtszeit- as for scientific journals, such as Nature and Science. The raum vermittelte die MDC-Pressestelle rund 500 Interviews Press Office also published eight press reports summarizing für Tageszeitungen, Magazine, Wochenzeitungen, Fernsehen, the coverage of the MDC and its collaborators. The MDC Hörfunk sowie für wissenschaftliche Magazine wie Nature Press Office wrote several contributions for various brochu- und Science, die sie inhaltlich und organisatorisch vorberei- res, including those for the Helmholtz Association, the tete. Darüber hinaus gab die Pressestelle in dem genannten Campus News of the BBB Management GmbH Berlin-Buch Zeitraum acht Pressespiegel heraus. Weiter verfasste die Campus, Partner for Berlin, as well as contributions to other MDC-Pressestelle Beiträge für verschiedene Broschüren, magazines and book projects. In addition, the MDC Press darunter für die Helmholtz-Gemeinschaft, die Campus News Office updated the pages on the Internet in German and der BBB Management GmbH Campus Berlin-Buch, Partner English which were adapted to reflect the new structure of the für Berlin, sowie für Buchprojekte. Zudem aktualisierte die program-oriented funding of the Helmholtz Association MDC-Pressestelle im Berichtszeitraum die von ihr zu verant- (HGF). wortenden Seiten des Internets in Deutsch und Englisch, die an die neue Struktur der programmorientierten Förderung der Since January 2003, the Press Office has published a bilin- Helmholtz-Gemeinschaft (HGF) angepasst wurden. gual, internal Newsletter “News in Brief” (“Kurz and Knapp”) at irregular intervals to inform the employees about Seit Januar 2003 gibt die Pressestelle darüber hinaus in unre- the most important developments at the MDC. gelmäßigen Abständen einen internen Newsletter „Kurz und Knapp“/„News in Brief“ heraus. Er informiert die Mitarbeite- During the 2002/2003 period, the MDC Press Office organi- rinnen und Mitarbeiter in Deutsch und Englisch über die zed a series of popular scientific Sunday lectures in the wichtigsten Entwicklungen im MDC. Berlin-Pankow Town Hall, started by the MDC in 1992. In addition, the MDC, along with the Berlin-Buch Campus, par- Im Rahmen der Öffentlichkeitsarbeit führte die MDC-Presse- ticipated in Berlin’s citywide “Long Night of the Sciences”. stelle auch 2002/2003 die Serie populärwissenschaftlicher Furthermore, the MDC Press Office hosted around 500 visi- Sonntagsvorlesungen im Rathaus Berlin-Pankow fort, die das tors, including students. MDC 1992, dem Jahr seiner Gründung, initiiert hatte. Darü- ber hinaus beteiligte sich das MDC mit dem Campus Berlin- Head: Barbara Bachtler Buch im Berichtszeitraum an der in ganz Berlin durchgeführ- ten „Langen Nacht der Wissenschaften“. Weiter betreute die MDC-Pressestelle 2002/2003 rund 500 Besucher, darunter Studenten und Schüler.

Leiterin: Barbara Bachtler 190

Administration Verwaltung

Personnel Personal

The MDC Personnel Department is responsible for all mat- Die Mitarbeiterinnen und Mitarbeiter dieser Abteilung sind ters relating to staff, wages, salaries, separation allowances, verantwortlich für den Abschluss von Arbeitsverträgen und staff hiring and removal, and travel expenses. Thus, the main alle damit im Zusammenhang stehenden Angelegenheiten. duty of our department is to support to all employees and Hauptanliegen der Personalabteilung ist die Betreuung aller guests via counselling and educational training. We pay spe- Mitarbeiter und Gäste des MDC durch Beratung und Fortbil- cial attention to our foreign employees. dungsangebote. Besonderes Augenmerk legen wir dabei auf unsere ausländischen Mitarbeiter. The MDC is currently financing 44 graduate students studying for a PhD. In addition, there are 69 part-time, third-party Gegenwärtig sind am MDC 44 Doktoranden angestellt. Da- financed junior scientists. Among the 337 total scientists, 74 rüber hinaus werden aus Drittmitteln 69 teilzeitbeschäftigte are foreigners representing 25 countries. As in the past, most Nachwuchswissenschaftler finanziert. Unter den 337 Wissen- (85 percent) of the scientists’ contracts are limited to a maxi- schaftlern kommen 74 aus 25 verschiedenen Ländern. Nach mum of five years. wie vor ist der Anteil der befristeten Verträge bei Wissen- schaftlern mit 85 Prozent sehr hoch. Additional statistics regarding the personnel status can be found in the following diagram. Die Angaben zum Personal der letzten Jahre und deren Finanzierung sind im folgenden Diagramm enthalten. Head: Dr. Hans-Joachim Seehrich Leiter: Dr. Hans-Joachim Seehrich

Personnel status. Distinctions according to financial sources Personal, unterschieden nach Finanzierung

800 763 739 693 692 154 175 700 652 653 144 140 159 144 600 169 154 154 162 500 145 148 Personnel status 97 400 Scientific Staff Wissenschaftler 65 67 82 (third-party funded) (Drittmittel-finanziert) 54 52 330 343 300 331 320 296 307 Scientific Staff Wissenschaftler (MDC-funded) (Haushalts-finanziert) 200 Technical/Admin. Staff Nicht-Wissenschaftler (third-party funded) (Drittmittel-finanziert) 100 Technical/Admin. Staff Nicht-Wissenschaftler (MDC-funded) (Haushalts-finanziert) 0 1998 1999 2000 2001 2002 2003 191

Finances Finanzen

The finance department concerns itself with all matters relat- Die Finanzabteilung befasst sich mit allen Angelegenheiten ing to the MDC’s financial funding, including accounting. der Einnahmen- und Ausgabenplanung des MDC, einschließ- The primary source (90%) of the MDC’s annual funding lich des Rechnungswesens. Es wurden die Voraussetzungen comes from the Federal government (Federal Ministry of geschaffen, dass die Kosten-Leistungs-Rechnung ab 2003 ge- Education and Research, BMBF) and the remaining 10% is nutzt werden kann, um eine transparente Kostendarstellung provided by the State of Berlin (Senate Administration for und -verursachung anzuwenden. Der Hauptanteil (90 %) der Science, Research and Culture). Within the framework of its jährlich verfügbaren finanziellen Mittel des MDC stammt aus basic funding, the MDC received over 52 million Euros for dem Bundeshaushalt (Bundesministerium für Bildung und the years 2002 and 2003. In addition, the MDC was able to Forschung). Die verbleibenden 10 Prozent werden vom Land increase its procurement of funds through third-party (exter- Berlin (Senatsverwaltung für Wissenschaft, Forschung und nal) financial sources from 13.2 million Euros in 2002 to 13.7 Kultur) geleistet. Im Berichtszeitraum 2002/2003 verfügte million Euros in 2003. das MDC über eine Grundfinanzierung von über 52 Millio- nen Euro. Die positive Entwicklung bei der Einwerbung von Head: Wolfgang Kühlewind Drittmitteln konnte fortgeführt werden. So war das MDC in der Lage, die Drittmittel von 13,2 Millionen Euro im Jahre 2002 auf 13,7 Millionen Euro im Jahre 2003 zu steigern.

Leiter: Wolfgang Kühlewind

MDC’s Extra Mural Funding (in thousands of o) Drittmittel des MDC (in tausend o)

16.000 14.340

14.000 13.810 13.147 Total

BMBF

12.000 11.230 10.707 10.614 DFG 10.000 EU

Foundationds

8.000 7.297

6.614 Industry 6.360 6.000 Others 4.192 3.997 3.756 3.535 3.582 3.398 4.000 3.393 3.272 3.370 2.374 1.819 2.000 1.509 1.115 1.289 1.010 1.079 1.011 864 1.052 876 786 862 695 780 468 543 376 553 297 385 457 420 462 0 1998 1999 2000 2001 2002 2003

Budget of the MDC (in thousands of o) Budget des MDC (in tausend o)

60000,00

20.056 50000,00 19.797 20.946 18.321 19.969 19.175 Expenditures for personnel 40000,00

14.517 Expenditures in 12.629 material assets 30000,00 12.542 12.207 14.055 12.431 Allocations for clinical research 20000,00 5.133 5.280 4.141 4.427 4.410 4.775 Investments 10000,00 14.945 11.478 14.258 16.428 16.952 12.829

0,00 1998 1999 2000 2001 2002 2003 (estimated) 192

Einkauf und Materialwirtschaft

Die Arbeit der Abteilung Einkauf und Materialwirtschaft des Max-Delbrück-Centrums für Molekulare Medizin (MDC) Berlin-Buch konzentrierte sich in den Jahren 2002 und 2003 auf drei Schwerpunkte:

• Rasche und bestmögliche Versorgung mit qualitätsgerech- ten Labor-, Hilfs- und Verbrauchsstoffen sowie Geräten unter Beachtung größtmöglicher Wirtschaftlichkeit • Schrittweise Einführung eines elektronischen Bestell- systems zur effektiven und transparenten Gestaltung der Einkaufsvorgänge • Überarbeitung und Neufassung der Beschaffungsordnung Säule Durchgang Torhaus Copyright: MDC/Photograph: Gunter Lepkowski Column of the Gatehouse Die schrittweise Einführung eines elektronischen Bestell- Copyright: MDC/Photographer: Gunter Lepkowski systems wird den Arbeitsaufwand der Mitarbeiter in der Abteilung entlasten. Nach Anschluss an das hausinterne Datenverarbeitungsnetz können die Mitarbeiter ihre Bestell- anforderung selbst eingeben. Damit ist im Einkauf eine schnellere Bearbeitung gewährleistet. Die Vernetzung der Arbeitsplätze der Wissenschaftler mit dem Einkauf ist auch eine wichtige Voraussetzung für die zukünftige Datenfern- Purchasing and Materials Management übertragung mit entsprechenden Labor- und Fachhändlern nach Abschluss von Rahmen- und Abrufverträgen. The work of the Purchasing and Materials Management Department is focused on three main areas: Leiter: Dr. Peter Konzer

• Rapid and efficient supply of quality laboratory materials and equipment at cost-effective rates • Step-by-step introduction of an electronic ordering department, enabling an effective and transparent purchasing process • Revision and compilation of new, up-to-date rules of procurement

The step-by-step introduction of an electronic ordering system will ease the workload of the department. After hooking up to the company's internal data processing network, employees will be able to log in and place their orders themselves. This will guarantee the fast processing of purchasing orders. Linking the scientists’ work places to the purchasing department is also an important requirement for the future transfer of data to contracted laboratory and spe- cialist suppliers.

Head: Dr. Peter Konzer 193

Central Facilities Zentrale Dienste

Library Bibliothek

The MDC library is a specialized scientific library involved Die Bibliothek des Max-Delbrück-Centrums für Molekulare in providing and supplying information in all research areas Medizin (MDC) Berlin-Buch ist eine moderne wissenschaft- of the MDC and its associated clinics. The library acts as a liche Spezialbibliothek, die die Forschung am MDC mit der modern information centre using advanced network struc- Bereitstellung von Literatur, Online-Informationsvermittlung tures and allowing access to information sources around the aus externen Datenbanken sowie der Lieferung von Informa- world. tionen in gedruckter und anderer Form unterstützt.

Modern information networks offer increased opportunities Sie arbeitet als eine Mischform aus Präsenzbibliothek mit to supply users with specific literature and information re- Freihandaufstellung und Ausleihe mit offenem Magazin. Der sources at their work place. The collection contains more than Gesamtbestand beträgt über 45 700 Medieneinheiten und 230 45,700 items and 230 print periodicals as well as different laufend gehaltene Periodica, zusätzlich über 1000 Titel in kinds of non-print materials. In addition to the print subscrip- elektronischer Form. Unter den wichtigsten Literaturdaten- tions, the library offers more than 1,000 electronic journals. banken sind Medline (Zugriff ab 1966, Web of Knowledge Campus-wide provision of major local databases include incl. Current Contents Connect (in 5 Ausgaben), Web of Medline (starting from 1966) and Web of Knowledge (includ- Science (Zugriff ab 1980) und der Science Citation Index. ing 5 editions of Current Contents Connect and Web of Science starting from 1980) and CD-Rom databases via inter- Der Freihandbereich verfügt über 26 Arbeitsplätze, 5 Compu- nal MDC-network with a range of scientific options. ter-Plätze mit Internet-Zugang und 3 Plätze, die einen laptop- Anschluss gestatten. Als lokales Bibliothekssystem arbeitet The open area provides 26 reading desks, 5 computer work- das Datenverwaltungs- und Informationssystem SISIS. Der stations with Internet access, and 3 access points for laptops. OPAC (Online Public Access Catalogue) verzeichnet den ge- The library operates with the local library computer system samten Bibliotheksbestand und ist über das Internet recher- SISIS. The OPAC (Online public access catalogue) lists all chierbar. Durch die Einbindung des Bibliotheksnetzes in das collections and is also available via the Internet. Printed cata- Campusnetz stehen alle elektronischen Informationsangebote logues describing older holdings with alphabetical and classi- campusweit zur Verfügung. Durch seine Client-Server-Archi- fied indices are also available while the library and its tektur gestattet das Bibliotheksnetz seinen Nutzern einen services are all available via the Internet. The client-server schnellen, oberflächenunabhängigen und direkten Zugriff zu architecture provides fast access, regardless of the user’s den aufliegenden Datenbanken. Über ihre Homepage präsen- operating system. The library offers a selection of main links tiert sich die Bibliothek im Internet, stellt dort eine Auswahl related to research on the campus. ihrer Dienste zur Verfügung und bietet eine Reihe dem For- schungsprofil angepaßter links. Head: Dr. Dorothea Busjahn Leiterin: Dr. Dorothea Busjahn 194

„Großes Sonnenzeichen I“, Bronze,1995, Rainer Kriester, vor dem MDC.C Copyright: MDC, Photograph: Gunter Lepkowski „Large Sun Drawing I“, Bronze,1995, Rainer Kriester, in front of the MDC.C Copyright: MDC, Photographer: Gunter Lepkowski

Animal Facilities Tierhaltung

Among the research institutes in Berlin, the MDC animal Unter den Forschungsinstituten Berlins besitzt die MDC-Ver- facility possesses the most genetically engineered rat and suchstierhaltung die meisten gentechnisch veränderten Rat- mice stocks. The four animal houses support transgenic and tenlinien und Mäusestämme. Mehr als 400 Knock-out-Stäm- animal experimentation at the MDC. Mice and rats are bred me sind gegenwärtig verfügbar als experimentelle Modelle in a disease-free environment. More than 400 strains of für die Erforschung von Krebs-, Herzkreislauf- und neurolo- knock-out mice are now available as experimental models in gischen Erkrankungen. Die 5 Tierhäuser des MDC versorgen cancer, cardiovascular, and neurological research. Encompas- die transgenen Versuchstiere und die dazugehörigen Experi- sing an area of 1,260 square-meters, the facilities include ani- mente. Mäuse und Ratten werden unter SPF-Bedingungen mal rooms (636 m2), surgery rooms, storage rooms, and cage gezüchtet und gehalten. Lokalisiert auf 1.260 Quadratmeter washing facilities. Nevertheless, the number of available Fläche, enthalten die Tierhäuser Tierräume (636 Quadratme- animal rooms and surgical facilities in and around the MDC ter), Labore, Lagerräume und Räume für die Käfigreinigung. does not meet present demands. Trotz dieser umfangreichen Flächen kann der Bedarf für die Wissenschaftler nicht erfüllt werden. The rapidly growing number of genetically engineered models of severe human illness means that further animal Die schnell wachsende Zahl genetisch modifizierter Modelle facilities for breeding and experimentation are needed. für verschiedene Humanerkrankungen bedeutet, dass weitere Therefore, the MDC is building a new central animal house. Tierhaltungsflächen zur Zucht und Haltung im Experiment This new facility will possess substantially more capacity for erforderlich sind. Deshalb hat das MDC ein neues zentrales animals (29 animal rooms covering a 1,015 m2 area) and Tierhaus gebaut, das im Laufe des Jahres 2004 fertiggestellt laboratories (19 labs over 665 m2) and will be opened during und bezogen wird. Die neue Tierhaltung wird substantiell the year 2004. mehr Kapazität für Tiere (29 Tierräume mit einer Fläche von 1.015 Quadratmetern) und Labore (19 Labore mit 665 Qua- Head: Dr. Karin Jacobi dratmetern Fläche) besitzen.

Leiterin: Dr. Karin Jacobi 195

Data and Image Processing EDV und Bildverarbeitung

The MDC Department of Data and Image Processing is re- Im MDC ist die Gruppe EDV und Bildverarbeitung verant- sponsible for the information technology (IT) deployment in wortlich für den IT-Einsatz in der Verwaltung und unterstützt the administration and supports the central scientific infra- die zentrale wissenschaftliche Infrastruktur. So betreut die structure. Thus, the Group takes care of the Central Server of Gruppe die zentralen Server des MDC und ermöglicht den the MDC and makes possible the use of electronic files, digi- Forschungsgruppen die Nutzung von File-, Backup-, Re- tal backups, remote access, and web services. The Group is moteAccess- und Web-Diensten. Ebenso ist die Gruppe also responsible for the Client-/Server operation of the verantwortlich für den Client-/Server-Betrieb der Verwaltung administration including the SAP System and user support. einschließlich der SAP-System- und -Nutzerbetreuung. 2003 The completed migration and expansion of the SAP system in wurde die Migration und Erweiterung des SAP-Systems 2003 was linked to the introduction of the cost and perfor- abgeschlossen, welche mit der Einführung der Kosten- und mance accounting and electronic purchasing systems. In the Leistungsrechnung und des Elektronischen Einkaufs verbun- future, we intend to strengthen the deployment of server- den ist. Künftig wollen wir den Einsatz Serverbasierter Tech- based technologies such as Citrix-Metaframe. Currently, the nologien wie Citrix-Metaframe verstärken. Gegenwärtig ist Group is working on the introduction of indexing services die Gruppe mit der Einführung von Verzeichnisdiensten und and the improvement of the electronic and data security der Verbesserung der Sicherheitslösung im MDC-Computer- within the MDC Computer Network. Netzwerk beschäftigt.

Head: Bernd Lemke Leiter: Bernd Lemke

Maintenance and General Services Haustechnik

The Department is in charge of the Appliance Service Groups Der Abteilung Haustechnik sind die Gruppe Geräteservice as well as all central glassware-cleaning rooms of the MDC. sowie sämtliche zentralen Spülküchen des MDC Berlin-Buch In the period of the report, the operation of the telephone unterstellt. Im Berichtszeitraum wurde die Bedienung der system was converted to WINDOWS 2000-oriented soft- Telefonanlage auf eine WINDOWS 2000-orientierte Soft- ware, enabling a very flexible operation independent of loca- ware umgestellt. Damit ist eine sehr flexible, vom Standort tion. A further achievement was the revision of the inter- unabhängige Bedienung, möglich. Ein weiterer Schwerpunkt linked fire alarm systems in the MDC buildings. Recently, war die Überarbeitung der vernetzten Brandmeldeanlagen in documentation of the MDC buildings has been drawn up and den MDC-Gebäuden. In der zurückliegenden Zeit wurde all areas recorded according to DIN [German Industrial gemeinsam mit der BBB Management GmbH Campus Ber- Standard] 227 jointly with the BBB Management GmbH lin-Buch unter dem Gesichtspunkt eines modernen „Facility Campus Berlin-Buch under the vision of modern “Facility Management“ eine Dokumentation der MDC-Gebäude erar- Management”. beitet und alle Flächen nach DIN 227 erfasst.

Head: Harry Schenk Leiter: Harry Schenk 196

Index Bhattacharya, Sarbani ...... 90 Bick-Sander, Anika ...... 152 Bienert, Ralf ...... 90 A Biese, Petra ...... 119 Abdel-Aty, Hassan ...... 40, 41 Bimmler, Marion ...... 184 Abdelaziz, Ihab ...... 43 Birchmeier, Carmen ...... 66, 67, 127, 128, 130, 131, Abdelilah-Seyfried, Salim ...... 13, 44, 45 ...... 133, 134, 137, 145, 156, 159 Aboujamra, Rami ...... 58 Birchmeier, Walter ...... 9, 59–61, 66, 67–69, Abraham, Claudia ...... 148 ...... 72, 133, 134, 157, 183, 184 Adams, Frauke ...... 29 Birkenfeld, Andreas ...... 29, 32 Ahlers, Annette ...... 79 Bit-Avragim, Nana ...... 45 Akepati, Vasudheva Reddy ...... 150 Blachut, Susanne ...... 21 Alenina, Natalia ...... 23 Blankenburg, Michaela ...... 96 Alexander, Christiane ...... 129, 149, 150 Blankenstein, Thomas ...... 64, 116, 117, 183, 184 Al-Kateb, Hussam ...... 24, 25 Blendinger, Gitta ...... 120, 121, 122 Al-Saadi, Nidal ...... 40, 41 Bochmann, Melanie ...... 41 Altmüller, Janine ...... 58 Bock, Petra ...... 41 An, Junfeng ...... 37 Böddrich, Annett ...... 148 Andersen, Olav ...... 17 Boden, Caroline ...... 69 Andrade, Miguel ...... 55 Boehm, Siegfried ...... 37, 92 André, Francoise ...... 58 Bohl, Steffen ...... 41 Anirudhan, Gireesh ...... 145 Böhm, Kerstin ...... 90 Anzenberger, Uwe ...... 17 Böhnke, Jana ...... 32 Arlt, Franziska ...... 72 Bommert, Kurt ...... 79, 85, 102, 103, 106, 107, 183, 184 Arnold, Susanne ...... 141 Bönsch, Regina ...... 145 Asbrand, Christian ...... 68 Bork, Peer ...... 52, 54, 55 Astrosini, Christian ...... 72 Borowiak, Malgorzata ...... 133 Aumann, Jutta ...... 72 Bortfeldt, Ralf ...... 55 Aydin, Atakan ...... 25 Boschmann, Michael ...... 28, 29, 32 Böttger, Adelheid ...... 23 Boye, Philipp ...... 41 B Braig, Melanie ...... 111 Babarovic, Paula ...... 74 Brandt, Moritz ...... 20, 152 Babich, Aleksei ...... 143 Breiderhoff, Tilman ...... 17 Babu, Harish ...... 152 Breitfeld, Dagmar ...... 100 Bachtler, Barbara ...... 189 Brembeck, Felix ...... 68 Bader, Michael ...... 13, 19, 22, 23, 43 Brinckmann, Anja ...... 58 Bähring, Sylvia ...... 21, 24, 25, 27, 28, 58 Britsch, Stefan ...... 130, 133, 134, 135, 137, 160 Baldinger, Tina ...... 85 Bröcker, Jana ...... 37, 159 Baltatu, Ovidiu ...... 23 Bröhl, Dominique ...... 131, 133 Barbosa, Marcos Eduardo ...... 23 Brohmann, Henning ...... 131, 133, 145 Bargou, Ralf C...... 102, 106, 107 Bröhmer, Meike ...... 79 Bartel, Sabine ...... 50 Brüning, Gerold ...... 152 Barthel, Denise ...... 117 Brylka, Heike ...... 135 Bartunek, Petr ...... 120, 121, 122 Buchert, Sven ...... 133 Bärwolff, Dieter ...... 94 Budziarek, Petra ...... 29 Bashammakh, Saleh ...... 23 Bulczak, Anita ...... 150 Bäßmann, Ingelore ...... 58 Burbach, Nadine ...... 74 Bauerfeind, Anja ...... 55 Burckle, Celine ...... 23 Baurand, Anthony ...... 39 Burmeister, Regina ...... 17 Bechstein, Josefine ...... 39 Buschow, Christian ...... 117 Becker, Christian ...... 58 Busjahn, Andreas ...... 24, 25, 53 Becker, Mareike ...... 87 Busjahn, Dorothea ...... 193 Becker, Michael ...... 126 Buttgereit, Jens ...... 23 Becker, Monika ...... 126 Bednorz, Carmen ...... 58 Begay, Valerie ...... 74 C Behrens, Diana ...... 126 Cajavec, Branka ...... 148 Ben Salem, Ali ...... 154 Calkhoven, Cornelis F...... 61, 74–76 Benga, Jemina ...... 100 Calließ, Christiane ...... 74, 76 Bengs, Marion ...... 48 Campos Cardoso, Cibele ...... 23 Bergmann, Martin W...... 38, 39, 160 Campos, Luciana Aparecida ...... 23 Bergmann, Nora ...... 52 Cardoso, Cristina M...... 25, 46–48, 78 Bergmann, Stephan ...... 72 Cayla, Cécile ...... 23 Betz, Regina ...... 58 Charo, Jehad ...... 117 197

Chatterjee, Manik ...... 102, 106, 107 Engelsberg, Arne ...... 105 Chmielowiec, Jolanta ...... 68 Enigk, Sabine ...... 58 Choi, Mira ...... 24, 25, 47, 78, 79 Entz, Patricia ...... 58 Christ, Baghat ...... 87 Essin, Kirill ...... 27 Cibrian, Elena ...... 45 Euler, Uta ...... 120, 122 Ciccarelli, Francesca ...... 55 Cichon, Günter ...... 118 Cifire, Felix ...... 100 F Cirak, Sebahattin ...... 58 Faelber, Katja ...... 90 Claßen, Elvira ...... 95, 96 Faerber, Katrin ...... 140 Clemens, Jan ...... 58 Falk, Kirsten ...... 64, 114, 123, 124 Cloos, Birgit ...... 90, 143 Faltysek, Maik ...... 148 Czerwony, Grit ...... 109 Feineis, Silke ...... 39 Feldpausch, Mareike ...... 32 Feller, Katharina ...... 68 D Ferreira, Anderson ...... 23 Däbritz, Henry ...... 111 Feske, Anette ...... 90 Dahm, Stefan ...... 55 Fesüs, Gabor ...... 27 Damm, Henning ...... 32 Fichtner, Iduna ...... 106, 125, 126, 183, 184 Daniel, Peter ...... 112, 113 Fiebeler, Anette...... 24, 25, 30 Das, Debashish ...... 143 Fiket, Maja ...... 150 Debs, Pierre ...... 85, 117 Fischer, Björn ...... 58 Dechend, Ralf ...... 24, 25, 49 Fischer, Heike ...... 58 Decker, Jochen ...... 145 Fischer, Judith ...... 21, 96 Del Barba, Marco ...... 150 Fischer, Uta ...... 119 Delbrück, Heinrich ...... 90 Forro, Gaby ...... 113 Dettmer, Rudolf ...... 79 Frahn, Anja ...... 186, 187 Diehl, Linda ...... 100 Franke, Gabriele ...... 28, 29, 32 Diester, Ilka ...... 58 Franke, Renate ...... 68 Dietz, Rainer ...... 25, 36–38, 40, 41, 49, 58 Frege, Renate ...... 95, 96 Dimitrova, Lora ...... 94 Freier, Janette ...... 50 Dobernack, Gisela ...... 43 Freund, Christian ...... 39, 79 Docherty, Neil ...... 23 Friede-Strauch, Manuela ...... 23 Doerks, Tobias ...... 55 Friedl, Sabine ...... 103, 106 Dokup, Kornelia ...... 58 Friedrich, Beate ...... 30 Domaing, Petra ...... 48 Friedrich, Matthias ...... 40, 41 Donath, Stefan ...... 37 Fritzsche, Anja ...... 148 Dörken, Bernd ...... 64, 78, 79, 85, 99–102, Fujita, Yasuyuki ...... 66, 68, 82, 133, 157 ...... 103, 105, 106, 109, 113, 115, 121 Fürst, Robert ...... 53, 54, 55 Drechsler, Hannelore ...... 143 Drenckhahn, Jörg ...... 34 Driessner, Madlen ...... 143 G Droese, Jana ...... 98–100, 105 Gahl, Anja ...... 48 Dröge, Anja ...... 148 Gaiser, Olaf ...... 90 Dubrovska, Galina ...... 27 Galle, Renate ...... 96 Duc Nguyen, Tuan ...... 114, 115 Gamper, Ivonne ...... 122 Dunken, Marianne ...... 109 Ganten, Detlev ...... 9, 18–21, 23, 58, 159, 177, 183 Ganzel, Karin ...... 79 Garcia, Ana ...... 152 E Garratt, Alistair ...... 128, 129, 131–134, 136, 137 Easwaran, Hariharan P...... 48 Gärtner, Angelika ...... 117 Ebert, Jan ...... 79 Gaertner, Klaus ...... 94 Eckert, Klaus ...... 126 Gast, Daniela ...... 152 Eckl, Christian ...... 57, 58 Gaudet, Francois ...... 47, 48 Eckl, Katja-Martina ...... 58 Gauss, Robert ...... 82 Ehninger, Dan ...... 152 Gedicke, Malenka ...... 58 Eigen, Marc ...... 17 Geibel, Sebastian ...... 100 Eilers, Andreas ...... 145 Geiler, Carola ...... 74 Ekici, Arif Bülent ...... 58 Geiss-Friedlander, Ruth ...... 82 El Jamali, Amina ...... 39, 79 Genser, Klaus ...... 148 Emmerich, Florian ...... 103 Georg, Bettina ...... 72 Engel, Carolin ...... 58 Gerlach, Brigitte ...... 140 Engel, Felix ...... 37 Gerull, Brenda ...... 34, 35, 159, 160 Engeli, Stefan ...... 28, 31, 32 Gibson, Meino ...... 140 Engels, Boris ...... 117–119, 159 Gierl, Mathias ...... 131, 133 198

Gillissen, Bernhard ...... 113 Hauck, Ludger ...... 37 Girresch, Bärbel ...... 140 Haucke, Lisa ...... 58 Gladow, Monika ...... 117, 118, 119 Haupt, Irene ...... 140 Glass, Rainer ...... 140 Hava, David ...... 45 Gögülü, Mehmet ...... 96 Heere, Petra ...... 45 Göhler, Heike ...... 148 Heibroch Petersen, Helle ...... 17 Goldbrich, Beate ...... 52 Heidemann, Antje ...... 140 Goldscheid, Eva ...... 124 Heinemann, Udo ...... 59, 60, 63, 86–88, 90, 183, 184 Golfier, Sven ...... 100 Heinze, Veronika ...... 55 Gollasch, Maik ...... 25–27, 31, 32, 159 Hemmati, Philipp ...... 113 Goncharenko, Mykola ...... 122 Henneke, Marco ...... 58 Gong, Maolian ...... 19, 21, 56, 58 Hennies, Hans Christian ...... 58 Gorsch, Jenny ...... 100 Henning, Mechthild ...... 143 Gorzelniak, Kerstin ...... 32 Heppenstall, Paul ...... 23, 133, 143, 145 Gösele, Claudia ...... 19, 20, 21 Herbst, Daniel ...... 58 Gossen, Manfred ...... 62, 63, 84, 85, 102 Herbst, Martin ...... 148 Gotthardt, Michael ...... 13, 51, 52, 156, 159 Herold, Diana ...... 27 Gottschalk, Iris ...... 29, 32 Herrmann, Alexander ...... 55 Gottschling, Karin ...... 133 Herrmann, Pia ...... 103, 107 Grabbert, Martina ...... 117, 119 Herwig, Susanne ...... 118 Gras, Christiane ...... 140 Heuser, Arnd ...... 35 Graschew, Georgi ...... 72 Heusser, Karsten ...... 29 Graßhoff, Peter ...... 100 Hieronymus, Thomas ...... 120–122 Grelle, Gerlinde ...... 148 Hikosaka, Akira ...... 87 Grieben, Marlies ...... 37 Hinz, Michael ...... 62, 79, 102, 103 Gries, Margret ...... 106, 107 Hinzmann, Maria ...... 95, 96 Griffel, Carola ...... 137 Hirsch, Christian ...... 82 Grigull, Sabine ...... 72 Hoffmann, Anja ...... 140 Grohmann, Maik ...... 23, 48 Hoffmann, Katrin ...... 58, 159 Gross, Volkmar ...... 24, 25 Hoffmann, Rosemarie ...... 109 Großmann, Katja ...... 68 Hofmann, Corinna ...... 119 Grothe, Daniela ...... 37 Hofmann, Wera ...... 95, 96 Grüger, Sabine ...... 25 Hofstätter, Maria ...... 124 Grün, Michael ...... 106, 107 Hohaus, Annette ...... 52 Grunewald, Ingrid ...... 48, 184 Homuth, Volker ...... 25, 49, 50 Grünwald, David ...... 48 Hönemann, Dirk ...... 106, 107 Grusser, Mandy ...... 58 Höpken, Uta E...... 100, 104, 105, 114, 183, 184 Güner, Dilek ...... 113 Höpner, Sabine ...... 124 Gunia, Wilfried ...... 55 Horn, Denise ...... 95, 96 Günther, Susanne ...... 122 Horn, Sabrina ...... 117 Gust, Tatjana C...... 121, 122 Horvat, Volker ...... 141 Hu, Jing ...... 145 Hübner, Norbert ...... 19, 20, 21, 52, 58, 134, 137, 156 H Hülsken, Jörg ...... 66–68, 157 Haas, Brigitte ...... 140 Hummel, Franziska ...... 103 Haase, Hannelore ...... 23, 43, 183, 184 Hummel, Kordelia ...... 119 Haase, Stephanie ...... 148 Hummel, Oliver ...... 20, 21 Hacker, Christine ...... 65, 79, 120–122 Haensch, Wolfgang ...... 71, 72 Hafez, Katrin ...... 55 I Hagena, Tina ...... 34 Iliescu, Radu ...... 23, 43 Haink, Petra ...... 122 Izsvák, Zsuzsanna ...... 87 Hakiy, Nahid ...... 117 Ivics, Zoltán ...... 62, 63, 86, 87 Halavaty, Andrei ...... 90 Hammes-Lewin, Annette ...... 17 Hampf, Mathias ...... 85 J Hänig, Christian ...... 148 Jacobi, Karin ...... 194 Hansmann, Georg ...... 37 Jäger, Katrin ...... 43 Happe-Kramer, Anna ...... 148 Jahnke, Jürgen ...... 32 Harjes, Phoebe ...... 148 Janczikowski, Marion ...... 50 Hartfuss, Eva ...... 45 Jandrig, Burkhard ...... 96 Härtig, Susann ...... 141 Janz, Martin ...... 103 Hartwig, Frauke ...... 32 Jarchow, Birgit ...... 140 Hasenjäger, Anne ...... 113 Jarosch, Ernst ...... 62, 82 Haßfeld, Sabine ...... 58 Jeblonski, Frank ...... 105 199

Jensen, Lars Juhl ...... 55 Kolbe, Michael ...... 90 Jerchow, Boris ...... 68 Kölble, Konrad ...... 96, 98 Jeske, Sunda ...... 115 König, Antje...... 113 Jeßberger, Sebastian ...... 152 Konzer, Peter ...... 184, 192 John, Anita ...... 126, 135 Köppen, Susanne ...... 148 Jordan, Jens ...... 25, 27–29, 32, 157, 160 Korbel, Jan ...... 55 Jorgas, Thorsten ...... 122 Koroll, Michael ...... 143 Joschko, Simone ...... 74 Kosel, Frauke ...... 68 Ju, Xin-Sheng ...... 122 Kostka, Susanne ...... 43, 148 Ju, Zhenyu ...... 58 Kotitschke, Erika ...... 152 Jundt, Franziska ...... 103 Kott, Monika ...... 23, 43 Junghahn, Ilse ...... 126 Kowenz-Leutz, Elisabeth ...... 74 Junghans, Christine ...... 25 Krahn, Inge...... 94 Jungmann, Sabine ...... 79 Krappmann, Daniel ...... 62, 78–80, 103, 160 Jüttner, René ...... 143 Krause, Julia ...... 58 Krause, Kerstin ...... 95, 96 Krause, Petra ...... 133 K Krella, Antje ...... 48 Kaada, Manuela ...... 43 Kreutz-Gers, Waltraud ...... 183 Kagelmaker, Horst ...... 140 Krivokharchenko, Alexander ...... 19, 23 Kahlem, Pascal ...... 111 Kronenberg, Golo ...... 128, 129, 152 Kalla, Claudia ...... 111 Krüger, Kerstin ...... 100, 103 Kamer, Ilona ...... 25 Krüger, Nadine ...... 29 Kammertöns, Thomas ...... 114, 117 Kuhle, Verona ...... 17, 87 Kanehl, Anke ...... 145 Kühlewind, Wolfgang ...... 191 Karawajew, Leonid ...... 106, 109 Kuhlmann, Grit ...... 186 Karczewski, Karin ...... 50 Kumar, Andreas ...... 40, 41 Karczewski, Peter ...... 43 Kuntzagk, Andreas ...... 55 Kärgel, Eva...... 79 Kupsch, Stefanie ...... 117 Kato, Lalitha ...... 21 Kurz, Steffen M...... 115, 120–122 Katzer, Andrea ...... 86, 87 Küttner, Irmgard ...... 117, 119 Kaufman, Chris ...... 87 Kaufmann, Lars ...... 154 Kausel, Tina ...... 148 L Keil, Claudia ...... 126 Lafuente, Dana ...... 23, 50 Kemmner, Wolfgang ...... 54, 71, 72 Lahnert, Peter ...... 94 Kempermann, Gerd ...... 128, 151, 152 Lalowski, Maciej ...... 148 Kempf, Hagen ...... 39 Lamounier-Zepter, Valéria ...... 23, 43 Kettenmann, Helmut ...... 127, 128, 129, 138, Lampert, Christoph ...... 117 ...... 140, 152, 183, 184 Langenickel, Thomas ...... 23 Kettritz, Ralph ...... 24, 25, 30, 46, 47, 78, 157, 159, 169 Lankisch, Katja ...... 111 Keyner, Daniela ...... 79, 100 Lapidous, Irina ...... 96 Khalina, Yana ...... 43 Lathe, Warren ...... 55 Khare, Dheeraj ...... 86, 87, 90 Lee, Soyoung ...... 110, 111 Kiesewetter, Hannes ...... 140 Lee, Young-Ae ...... 20, 21, 58, 157, 158 Kim, Hye-Jung...... 115, 117 Lehmann, Christine ...... 94 Kirsch, Ralf D...... 27, 120–122 Lehmann, Insa ...... 105 Kirst, Elisabeth ...... 58 Lehmann, Katarina ...... 58 Kistel, Heide ...... 19, 21 Lehmann, Kathrin ...... 152 Klahn, Sylvia ...... 82, 117 Lehmann, Mandy ...... 58 Klaus, Alexandra ...... 68 Leistner, Werner ...... 92 Klein, Eireen...... 25 Lemke, Bernd ...... 184, 194, 195 Kleinewietfeld, Markus ...... 124 Lemke, Britt ...... 122 Kleißle, Sabrina ...... 124 Lemm, Margit ...... 126 Klewer, Jacqueline ...... 45 Leng, Corinna ...... 114, 115 Kley, Katarina ...... 101, 103 Lentzsch, Suzanne ...... 106, 107 Knauf, Felix ...... 27 Lenzen, Kirsten ...... 58 Knaus, Thomas ...... 37 Leonhardt, Heinrich ...... 46–48 Knespel, Andreas ...... 90, 162 Leschke, Andrea ...... 133 Knespel, Siegne ...... 87, 120–122 Leutz, Achim ...... 60, 61, 73–76 Knoblich, Maria ...... 74 Lewin, Gary R...... 23, 129, 130, 133, 143–145 Koh, Ki-Ryang ...... 122 Li, Jincheng ...... 37 Köhler, Matthias ...... 30 Li, Li ...... 68 Köhler, Niklas ...... 118 Li, Liang-Ping ...... 117 Köhler, Tino...... 58 Li, Peifeng ...... 37 200

Liebner, Iska ...... 150 Monti, Jan ...... 20, 21, 52 Lier, Simone ...... 131, 133 Morano, Ingo ...... 23, 42, 43 Lietz, Andreas ...... 101–103 Mordmüller, Benjamin ...... 79 Lin, Shaoqiang ...... 72 Moré, Margret ...... 143, 183, 184 Lipp, Martin ...... 37, 59, 60, 97, 100, 114, 140, 183, 184 Morkel, Markus ...... 67–69 Lips, Janet ...... 37 Moshourab, Rabih ...... 145 Litscher, Dagmar ...... 148 Mühl, Astrid ...... 25 Liu, Weimin ...... 85 Müller, Anita ...... 19, 21 Lokareddy, Ravi Kumar ...... 90 Müller, Antje ...... 113 Lorenz, Susanne ...... 58, 113 Müller, Christine ...... 74, 75 Löser, Peter ...... 118 Müller, Claudius ...... 145 Lucius, Hans ...... 55 Müller, Dominik N...... 24, 25, 49, 159 Lucke, Barbara ...... 58 Müller, Eva-Christina ...... 148 Ludwig, Wolf-Dieter ...... 108, 109 Müller, Gerd ...... 100 Luft, Friedrich C...... 13, 14, 17, 19, 21, 24, 25, 27–30, 32, Müller, Jürgen J...... 90 ...... 47, 49, 50, 53, 54, 58, 78, 159, 183, 184 Müller, Thomas ...... 131, 133, 145, 184 Luganskaja, Tatjana ...... 55 Munz, Barbara ...... 68, 170 Lusatis, Simone ...... 115 Muschiol, Sandra ...... 58 Lustig, Barbara ...... 72 Lutsch, Gudrun ...... 23, 42, 152 Lutter, Steffen ...... 43 N Lützkendorf, Susanne ...... 69 Nätebus, Marc Oliver ...... 58 Nentwig, Brigitte ...... 30, 83 Neuber, Oliver ...... 82 M Neuhaus, Yvette ...... 25 Mácha, Inés ...... 58 Neumann, Claudia ...... 126 Mahiny, Azita ...... 100 Nguyen-Hoai, Tam ...... 115 Mai, Angelika ...... 141 Niewöhner, Jörg ...... 153, 154 Mailer, Reiner ...... 124 Nitschke, Ute ...... 103 Manjasetty, Babu A...... 89, 90 Nitz, Monika ...... 23 Mapara, Markus ...... 106, 114, 115 Nolte, Christiane ...... 140 Marenholz, Ingo ...... 58 Normand, Guillaume ...... 113 Marin Esteban, Viviana...... 123, 124 Nothnagel, Anita ...... 55 Margot, Jean B...... 48 Nothnagel, Michael ...... 55 Markovic, Darko ...... 140 Nowak, Danny ...... 48 Martin, Mathilde ...... 109 Nürnberg, Gudrun ...... 55 Martin, Nico ...... 58 Nürnberg, Peter ...... 13, 14, 21, 53, 56, 58 Martin, Robert ...... 48 Martinez de la Torre, Margaret ...... 133 Martinez-Salgado, Carlos ...... 145 O Mathas, Stefan ...... 79, 101–103, 107 Oancea, Ionela ...... 55 Matthes, Eckhart ...... 93, 94 Oeckinghaus, Andrea ...... 80 Max, Klaas ...... 90 Oltersdorf, Doreen ...... 109 Mehling, Heidrun ...... 29 Osterziel, Karl-Josef ...... 13, 14 Mehrhof, Felix ...... 37, 38 Otto, Albrecht ...... 17, 26, 37, 114, 148 Mering, Christian von ...... 55 Özcelik, Cemil ...... 131–134, 137 Merino, Vanessa Ferreira ...... 23 Meußer, Birgit ...... 82 Meyer, Birgit ...... 58 P Meyer, Dagmar ...... 100 Pachale, Dietlind ...... 58 Meyer, Philippe ...... 154 Panek, Anna ...... 23 Michely, Beate ...... 35 Pankow, Harald ...... 55 Mieth, Maren ...... 85 Pannasch, Ulrike ...... 140 Milan, Sigrid ...... 34 Pannek, Anja ...... 83 Milenkovic, Nevena ...... 145 Papst, Marion ...... 85 Mintzlaff, Sascha ...... 148 Patzke, Christopher ...... 143 Mirossay, Andrej ...... 111 Paul, Norbert ...... 154 Miskey, Csaba ...... 87 Pech, Jenny ...... 58 Mo, Xianming ...... 74 Perez-Iracheta, Carolina ...... 55 Mödig, Henriette ...... 74 Peter, Heidrun ...... 119 Mohebbi, Nilufar ...... 27 Peter, Jens-Uwe ...... 23 Möhring, Regina ...... 185 Petsch, Kerstin ...... 124 Mokros, Thilo ...... 100, 104, 105 Petzhold, Daria ...... 43 Moldenhauer, Ferdinand ...... 55 Pezzutto, Antonio ...... 64, 65, 114, 115, 121 Möller, Heinz ...... 68 Phillips, Andreas ...... 140 201

Pierschalek, Petra ...... 43 Rohr, Stefan ...... 45 Pietschmann, Andrea ...... 95, 96 Rohs, Remo ...... 91, 92 Pils, Birgit ...... 55 Rolfs, Frank ...... 152 Pinto, Dalila ...... 58 Rolle, Susanne ...... 25, 47, 78 Pisch, Erika ...... 148 Römer, Benedikt ...... 152 Plaumann, Marlies ...... 95, 96 Rosário, Marta ...... 68 Pless, Ole ...... 74 Rose, Jürgen ...... 55 Podlatis, Patricia ...... 122 Roske, Yvette ...... 90 Pohl, Bärbel ...... 39 Rossius, Jana ...... 113 Popova, Elena ...... 19, 23 Rötzschke, Olaf ...... 64, 114, 123, 124 Poppe, Brunhilde ...... 55 Rücker, Karin ...... 96 Poppe, Konstanze ...... 96 Rüder, Constantin ...... 105 Pospiech, Regina ...... 58 Rudolph, Carthrin ...... 133 Pospisil, Heike ...... 55 Rüschendorf, Franz ...... 55 Preiß, Susanne ...... 117 Ruf, Nico ...... 58 Puentes, Fabiola ...... 123, 124 Ruf, Rainer ...... 58 Punnoose, Alan ...... 37

S Q Saar, Kathrin ...... 58 Qin, Fu ...... 50 Sakamoto, Hideki ...... 48 Qin, Zhihai ...... 117 Sander, Thomas ...... 58 Quandt, Andreas ...... 58 Santibanez-Koref, Mauro ...... 55 Quensel, Christina ...... 30 Sasse, Verena ...... 135 Sasse-Klaassen, Sabine ...... 34, 35 Saul, Vera ...... 23 R Schabath, Richard ...... 109 Räbel, Katrin ...... 52 Shagdarsuren, Erdenechimeg...... 25 Raddatz, Katy ...... 52 Schäfer, Michael...... 143 Räder, Charlotte ...... 17 Schäffer, Hans-Jörg ...... 68 Radetzki, Silke ...... 113 Schäffer, Susanne...... 143 Radke, Michael ...... 52 Schalow, Tanja...... 133 Rahn, Peter ...... 100 Schaeper, Ute...... 68 Raible, Florian ...... 55 Scharschmidt, Erika...... 79, 80 Ramadanova, Kalina ...... 34 Scheele, Sylvia...... 113 Ranjan, Anand ...... 85 Scheer, Helga...... 94 Rasheed, Ata-Ur ...... 100 Scheidereit, Claus...... 25, 47, 62, 77–80, 101–103 Ratei, Richard ...... 109 Scheller, Marina...... 74 Rathee, Parvinder ...... 145 Schenk, Harry...... 195 Rathjen, Fritz G...... 129, 142, 143 Scherneck, Siegfried ...... 63, 64, 95, 96 Rautenberg, Susanne ...... 148 Schicktanz, Silke ...... 153, 154 Rechner, Cindy ...... 39 Schildhauer, Ines...... 111 Rehm, Armin ...... 105, 109, 110, 160 Schildt, Jürgen ...... 94 Reich, Jens G...... 11, 25, 53–55 Schipke, Carola...... 140 Reiche, Juliane ...... 17 Schlag, Peter M...... 54, 61, 69–72, 95, 96, Reif, Susanne ...... 154 ...... 113, 114, 159, 184 Reimann, Maurice ...... 111 Schlegel, Wolfgang-Peter ...... 43 Reimer, Tatiana ...... 105 Schmetzer, Oliver...... 114, 115 Reiterer, Philipp ...... 100 Schmidt, Christina ...... 137 Rejman-Lipinski, Anette ...... 96 Schmidt, Elena ...... 58 Rentzsch, Brit ...... 23 Schmidt, Hannes ...... 118, 143 Reuß, Simone ...... 119 Schmidt, Sabine ...... 21 Reuter, Katja ...... 133, 152 Schmidt, Susanne ...... 58 Rhein, Peter ...... 109 Schmidt, Steffen ...... 55 Richter, Anja ...... 113 Schmidt, Tanja ...... 30 Richter, Antje ...... 113 Schmidt-Ullrich, Ruth ...... 38, 78, 79, 184 Richter, Gerhild ...... 68, 115 Schmitt, Clemens A...... 110, 111, 159 Richter, Stefan ...... 109 Schmidt, Willy ...... 55 Ried, Christian ...... 100 Schmollinger, Jan ...... 117 Ritter, Heide ...... 58 Schnitzler, Mark ...... 115 Rocca, Elena ...... 137 Schober, Daniel ...... 55 Rodriguez Aznar, Eva ...... 143 Scholz, Christian ...... 113 Röefzaad, Claudia ...... 72 Schönherr, Anke ...... 148 Rohde, Elvira ...... 133 Schories, Barbara ...... 85, 103 Rohde, Klaus ...... 55 Schröder, Christoph ...... 28, 29 202

Schröder, Dirk ...... 121, 122 Straub, Petra ...... 117 Schugardt, Nancy ...... 148 Strauss, Anke ...... 29, 32 Schüler, Thomas ...... 117, 118 Strehle, Michael ...... 133 Schulz, Hannelore ...... 17 Strissel, Jana ...... 95, 96 Schulz, Herbert ...... 20, 21 Strobach, Stefanie...... 122 Schulz, Sabrina ...... 58 Strödicke, Martin ...... 148 Schulz, Sonja ...... 83 Struk, Bertold ...... 34 Schulz, Sylvia ...... 126 Stühmer, Torsten ...... 107 Schulze, Tobias ...... 100 Stulich, Marianne ...... 94 Schulze, Wolfgang ...... 50 Sturm, Isrid ...... 113 Schulze-Pröbsting, Kristina ...... 101–103 Suk, Eun-Kyung ...... 58 Schulz-Menger, Jeanette ...... 40, 41 Sümnich, Ingeborg ...... 95, 96 Schümann, Margitta ...... 148 Sun, Yinyan ...... 19–21 Schumi, Christian ...... 58 Sunami, Yoshiaki ...... 79 Schunk, Wolf-Hagen ...... 24, 26 Suopanki, Jaana ...... 148 Schütz, Gunnar ...... 68 Suyama, Mikita ...... 55 Schütz, Susanne ...... 17 Synowitz, Michael ...... 140 Schuster, Stefan ...... 55 Szangolies, Inka ...... 58 Schwabe, Holger ...... 70, 72 Szczech, Elke ...... 28, 29 Schwartz, Arnfried ...... 95, 96 Schwartze, Stefan ...... 183 Schwarz, Monika ...... 45, 58, 114, 115 T Schwarz-Romond, Thomas ...... 66, 68, 134 Tabor, Daniela ...... 115 Schweda, Mark ...... 154 Tank, Jens ...... 27–29, 32 Schwede, Heike ...... 100 Tannert, Christof ...... 153, 154 Seeger, Michaela ...... 140 Tauer, Ulrike ...... 58 Seehrich, Hans-Joachim ...... 190 Tegethoff, Sebastian ...... 78, 79 Segner, Ruth ...... 152 Teichert, Carsten ...... 27 Seifert, Alexandra ...... 145 Teichmann, Bianca ...... 111 Seifert, Wenke ...... 58 Tepke, Anne-Dorothe ...... 126 Seipold, Sabine ...... 45 Thiede, Jana ...... 58 Seitz, Susanne ...... 95, 96 Thiele, Holger ...... 55, 58 Selders, Lisa ...... 126 Thiele, Verena ...... 55 Sha, Xiaojin ...... 74 Thierfelder, Ludwig ...... 11, 13, 33–35, 51, 188 Shah, Parantu ...... 55 Thirunarayanan, Nanthakumar ...... 100 Shagdarsuren, Erdenechimeg ...... 25 Thränhardt, Heike ...... 145 Sharma, Arya M...... 25, 27–29, 31, 32 Thun, Irene ...... 152 Shin, Jung-Bum ...... 145, 159 Todiras, Mihail ...... 23 Shmidt, Tanja ...... 23 Toksöz, Engin ...... 148 Sieber, Martin ...... 133 Toliat, Mohammad Reza ...... 58 Siele, Dagmar ...... 95, 96 Trippmacher, Ilona ...... 35 Simon, Bernd ...... 55 Tschapek, Alexej ...... 55 Simon, Judith...... 154 Tünnemann, Gilla ...... 48 Sipo, Isaac ...... 100 Tünnemann, Gisela ...... 90 Sklenar, Gisela ...... 90 Turnbull, Andrew P...... 90 Sklenar, Heinz ...... 63, 91, 92 Skorna, Madeleine ...... 58 Smink, Jeske ...... 74 U Smith, Janice ...... 72 Uckert, Wolfgang ...... 64, 65, 118, 157, 158, 184 Sochnikova, Natalia ...... 68 Uhlenberg, Birgit ...... 58 Söderhall, Cilla ...... 58 Uhlmann, Karen ...... 187 Sommer, Thomas ...... 62, 66, 68, 81–83, 182 Uhlmann, Regina ...... 25 Sommermeyer, Daniel ...... 119 Unsöld, Thomas ...... 143 Specowiak, Tanja ...... 117 Utz, Wolfgang ...... 41 Spoelgen, Robert ...... 17 Sporbert, Anje ...... 48 Stade, Katrin ...... 83, 160, 184 V Stärck, Lilian ...... 113 van der Meer, Nico ...... 41 Starke, Mireille ...... 124 Vassioutina, Elena ...... 133 Stein, Ulrike ...... 70–72, 85 Verdoodt, Berlinda ...... 113 Steiner, Barbara ...... 152 Vergopoulos, Athanasios ...... 55 Stelzl, Ulrich ...... 148 Verlohren, Stefan...... 27 Stoeter, Michael ...... 41 Vetter, Donate ...... 17 Stoffels, Grit ...... 29 Vierich, Sandra ...... 109 Stollenwerk, Anke ...... 39 Vilianovitch, Larissa ...... 23 203

Vogel, Regina ...... 68 X Volkwein, Corinna ...... 82, 83 Xu, Hong ...... 74 von Haefen, Clarissa ...... 113 Xu, Yaxin ...... 21 von Harsdorf, Rüdiger ...... 37, 38, 158 von Janta-Lipinski, Martin ...... 94 von Kries, Jens-Peter ...... 68 Z Zacharias, Ute ...... 143 Zagrosek, Anja ...... 40, 41 W Zaitseva, Ioulia ...... 19, 23 Walisko, Oliver ...... 87 Zaragoza, Katrin ...... 74 Wallukat, Gerd ...... 13, 25, 49, 50, 159, 184 Zastrow, Inga ...... 55 Wälter, Stephanie ...... 148 Zayed, Hatem ...... 86, 87 Walther, Diego ...... 23 Zdobnov, Evgeny ...... 55 Walther, Ingrid ...... 68, 133 Zechner, Dietmar ...... 66, 68, 82, 133 Walther, Wolfgang ...... 70–72, 85, 126 Zehlike, Ulrike ...... 154 Wang, Liping ...... 140 Zeidler, Helga ...... 95, 96 Wanker, Erich ...... 128, 130, 146, 148 Zeisig, Reiner ...... 126 Waßmuth, Peter ...... 95, 96 Zeller, Constanze ...... 95, 96 Wassmuth, Ralf ...... 41 Zenke, Martin ...... 65, 79, 115, 120–122 Wegener, Elmar ...... 78–80 Zenkner, Martina ...... 148 Wegener, Monika ...... 50 Ziebold, Ulrike S...... 69 Wegner, Anja ...... 145 Ziegenhorn, Katja ...... 111 Weinert, Steffi ...... 52 Ziegler, Ulrike ...... 152 Weixler, Silke ...... 58 Zimdahl, Heike ...... 20, 21 Wellner, Maren ...... 17, 32, 47, 78 Zornik, Denise ...... 58 Welsch, Gabi ...... 39 Wende, Hagen ...... 131, 133 Wendt, Jana ...... 113 Wengner, Antje ...... 100 Wenzel, Katrin ...... 95, 96 Werner, Erik ...... 90 Werner, Sabine ...... 95, 96 Westen, Christel ...... 117, 184 Westermann, Jörg ...... 114, 115, 121 Wetzel, Christiane ...... 145 Wetzel, Katarina ...... 43 Wiedemann, Peter ...... 153, 154 Wiesenthal, Volker ...... 74, 76 Wilde, Frank ...... 100 Wildner, Hendrik ...... 131, 133 Wilhelm, Claudia ...... 23 Wilhelm, Sabine ...... 68 Willebrand, Ralf ...... 117 Willimsky, Gerald ...... 117 Willnow, Thomas E...... 11–13, 15, 17 Winkler, Liselotte ...... 23 Wirsching, Manuela ...... 58 Wissler, Susanne ...... 25, 29, 32 Witt, Christina ...... 109 Wittstruck, Angelika ...... 82 Wittstruck, Nadine ...... 58 Wiznerowicz, Irmgard ...... 68 Woischwill, Christiane ...... 43 Wolf, Edelgard ...... 55 Wolf, Matthias ...... 58 Wolf, Susanne ...... 152 Wollert-Wulf, Brigitte ...... 103 Worm, Uwe ...... 148 Wu, Xiaoqing ...... 119 Wu, Yong ...... 94 Wulf, Annika ...... 126 Wüstner, Daniel ...... 91, 92 204

April 2004